Method for producing polymer, method for producing organic acid, and organic acid-producing microorganism

ABSTRACT

The present invention provides a method of producing a polymer, which comprises the step of performing a polymerization reaction using, as a starting material, an organic acid obtained by allowing a microorganism or a treated cell thereof to act on an organic raw material, wherein said microorganism has an ability to produce an organic acid and has been modified so as to produce less aromatic carboxylic acid as compared to an unmodified strain.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/030,486 filed Sep. 18, 2013, allowed, which is a continuation ofInternational Application PCT/JP2012/056935, filed on Mar. 16, 2012, anddesignated the U.S., (and claims priority from Japanese PatentApplication 2011-061412 which was filed on Mar. 18, 2011), the entirecontents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method of producing a high-qualitypolymer with less coloration, a method of producing an organic acid forobtaining the polymer, an organic acid-producing microorganism used toproduce the organic acid.

BACKGROUND ART

Biodegradable plastic materials, which are eventually decomposed intowater and carbon dioxide by microorganisms, are used in a wide varietyof applications such as food containers and agricultural materials.

At present, such polyesters are produced by polycondensation of amaterial derived from a fossil fuel resource; however, in view of recentenvironmental problems at a global scale such as concerns for depletionof fossil fuel resources and increase in the atmospheric carbon dioxidelevel, attention has been drawn upon a method of deriving a startingmaterials of these polymers from a biomass resource.

So far, there have been disclosed technologies for producing adicarboxylic acid used as a starting material of a polyester, such assuccinic acid or adipic acid, from glucose, sucrose or the like of abiomass resource origin by a fermentation method (see Patent Documents 1and 2 and Non-patent Document 1).

In cases where a dicarboxylic acid is used as a starting material of apolymer, in order to maintain the polymerization activity and to therebyobtain a high-quality polymer with less coloration, a highly puredicarboxylic acid is required. As a method of purifying a dicarboxylicacid produced by a fermentation method, there are disclosed, forexample, a method in which an ion-exchange resin is used and a method inwhich electrodialysis is used (Patent Documents 3 and 4). Examples of asubstance which causes coloration of a polymer include those impuritiesthat exhibit absorption in the ultraviolet region of 250 to 300 nm, andit is described to be useful that such impurities be reduced to nothigher than a specific amount (Patent Document 5).

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: JP 11-113588A-   Patent Document 2: JP 11-196888A-   Patent Document 3: U.S. Pat. No. 6,284,904-   Patent Document 4: JP 2-283289A-   Patent Document 5: JP 2010-100617A

Non-Patent Documents

-   Non-patent Document 1: Journal of the American Chemical Society No.    116 (1994), 399-400

SUMMARY OF THE INVENTION

However, in these production methods described in Patent Documents 1 and2 and Non-patent Document 1, the resulting acid compound may contain avariety of impurities such as organic acids other than the desireddicarboxylic acid that were produced as by-products, sugars that wereleft without being completely assimilated by a microorganism, compoundscontaining elemental nitrogen originated from a biomass resource andmetal cations; therefore, a further improvement is necessary.

Furthermore, also in the dicarboxylic acids obtained by the purificationprocesses according to Patent Documents 3 and 4, when the dicarboxylicacids are used as a starting material of a polymer, for example,coloration tends to occur in the resulting polymer; therefore, a furtherimprovement is necessary.

Moreover, in Patent Document 5, the impurities causing coloration of apolymer were not identified and, from the practical standpoint, it is animportant problem to attain removal of such impurities from adicarboxylic acid by a more efficient and inexpensive method.

In view of the above, objects of the present invention are: to provide amethod of producing a high-quality polymer with less coloration; toprovide a method of producing an organic acid suitable for this purpose;and to provide a microorganism used to produce the organic acid.

In order to solve the above-described problems, the present inventorsintensively studied and discovered that, among a variety of impuritiesthat may be contained in an organic acid used as a starting material ofa polymer, aromatic carboxylic acids such as protocatechuic acid are thesubstances that cause coloration of a polymer; and that aromaticcarboxylic acids such as protocatechuic acid are not easily separated bypurification. Furthermore, the present inventors produced an organicacid by allowing a microorganism modified so that the production ofaromatic carboxylic acid such as protocatechuic acid is reduced, or atreated cell thereof, to act on an organic raw material and discoveredthat, by using such an organic acid to synthesize a polymer, colorationin the resulting polymer can be reduced, there by completed the presentinvention.

That is, according to the present invention, the following inventionsare provided.

[1] A method of producing a polymer, which comprises the step ofperforming a polymerization reaction using, as a starting material, anorganic acid obtained by allowing a microorganism or a treated cell ofthereof to act on an organic raw material, wherein said microorganismhas an ability to produce the organic acid and has been modified so asto produce less aromatic carboxylic acid as compared to an unmodifiedstrain.[2] The method according to [1], wherein said microorganism has beenmodified so that at least one enzyme activity selected from the groupconsisting of DAHP synthase activity, dehydroquinate synthase activity,dehydroquinate dehydratase activity and dehydroshikimate dehydrataseactivity is reduced as compared to an unmodified strain, and productionof an aromatic carboxylic acid is thereby reduced.[3] The method according to [1] or [2], wherein said organic acid issubjected to a crystallization treatment.[4] The method according to any one of [1] to [3], wherein said aromaticcarboxylic acid is a hydroxybenzene carboxylic acid.[5] The method according to any one of [1] to [4], wherein said organicacid is succinic acid.[6] The method according to [5], wherein said polymer is a polyester ora polyamide.[7] A method of producing an organic acid, comprising the step ofallowing a microorganism or a treated cell thereof to act on an organicraw material, wherein said microorganism has an ability to produce theorganic acid and has been modified so as to produce less aromaticcarboxylic acid as compared to an unmodified strain.[8] The method according to [7], wherein said microorganism has beenmodified so that at least one enzyme activity selected from the groupconsisting of DAHP synthase activity, dehydroquinate synthase activity,dehydroquinate dehydratase activity and dehydroshikimate dehydrataseactivity is reduced as compared to an unmodified strain, and productionof an aromatic carboxylic acid is thereby reduced.[9] The method according to [7] or [8], wherein said microorganism or atreated cell thereof is allowed to act on said organic raw material inan anaerobic atmosphere.[10] The method according to any one of [7] to [9], wherein said organicacid is succinic acid.[11] The method according to any one of [7] to [10], further comprisingthe step of performing a crystallization treatment of said organic acid.[12] The method according to any one of [7] to [11], wherein saidaromatic carboxylic acid is a hydroxybenzene carboxylic acid.[13] The method according to any one of [7] to [12], wherein saidmicroorganism is at least one bacterium selected from the groupconsisting of coryneform bacteria, bacteria belonging to the genusMycobacterium, bacteria belonging to the genus Rhodococcus, bacteriabelonging to the genus Nocardia and bacteria belonging to the genusStreptomyces.[14] A coryneform bacterium, which has an ability to produce an organicacid and has been modified so that at least one enzyme activity selectedfrom the group consisting of dehydroquinate synthase activity,dehydroquinate dehydratase activity and dehydroshikimate dehydrataseactivity is reduced as compared to an unmodified strain, and theproduction of an aromatic carboxylic acid is thereby reduced.[15] The coryneform bacterium according to [14], which has been modifiedso that the dehydroshikimate dehydratase activity is reduced.[16] The coryneform bacterium according to [15], wherein saiddehydroshikimate dehydratase activity is reduced by disrupting a geneencoding dehydroshikimate dehydratase or by introducing a mutation intosaid gene.[17] The coryneform bacterium according to [16], wherein said geneencoding dehydroshikimate dehydratase is a DNA comprising the nucleotidesequence shown in SEQ ID NO:15 or a DNA which hybridizes with acomplementary sequence of said nucleotide sequence shown in SEQ ID NO:15under stringent conditions and encodes a protein having dehydroshikimatedehydratase activity.[18] The coryneform bacterium according to any one of [14] to [17],wherein said aromatic carboxylic acid is a hydroxybenzene carboxylicacid.[19] The coryneform bacterium according to [18], wherein said aromaticcarboxylic acid is protocatechuic acid.

By the present invention, production of aromatic carboxylic acids whichcause coloration of a polymer can be reduced, so that, by using theorganic acid of the present invention as a starting material of apolymer, a high-quality polymer with less coloration can be obtained. Bythe present invention, the purification step of an organic acid can besimplified and the production cost can thus be reduced. Furthermore, thepresent invention can greatly contribute to solving the environmentalproblems and problems of depletion in fossil fuel resources and thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the synthetic pathway of protocatechuicacid.

FIG. 2 is a diagram illustrating the procedures for constructing aplasmid pMJPC17.2. The underlined numbers indicate the primers havingthe sequence shown in the corresponding SEQ ID NOs.

FIG. 3 is a diagram illustrating the procedures for constructing aplasmid pQsuB1.

FIG. 4 is a drawing of a crystallization apparatus.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The embodiments of the present invention will now be described indetail.

<Microorganism of the Present Invention>

The microorganism according to the present invention has an ability toproduce an organic acid and has been modified so as to produce lessaromatic carboxylic acid as compared to an unmodified strain. Theexpression “modified so as to produce less aromatic carboxylic acid ascompared to an unmodified strain” encompasses a condition in which theactivity of an enzyme relating to a biosynthetic pathway of an aromaticcarboxylic acid is directly and indirectly reduced as compared to anunmodified strain; a condition in which, because of an increase in theactivity of an enzyme of other biosynthetic pathway which shares aprecursor with an aromatic carboxylic acid but branches from thebiosynthetic pathway thereof, the flow of the precursor of the aromaticcarboxylic acid to its biosynthetic pathway is reduced, so that thebiosynthesis of the aromatic carboxylic acid is indirectly reduced; anda condition in which the activity of an enzyme relating to adecomposition pathway of an aromatic carboxylic acid is directly andindirectly increased as compared to an unmodified strain.

Specific examples of such microorganism include those which have beenmodified so that at least one enzyme activity selected from the groupconsisting of DAHP synthase activity, dehydroquinate synthase activity,dehydroquinate dehydratase activity and dehydroshikimate dehydrataseactivity is reduced as compared to an unmodified strain. Thereamong, themicroorganism of the present invention is preferably a microorganismwhich has been modified so that at least one enzyme activity selectedfrom the group consisting of dehydroquinate synthase activity,dehydroquinate dehydratase activity and dehydroshikimate dehydrataseactivity is reduced as compared to an unmodified strain, more preferablya microorganism which has been modified so that dehydroquinatedehydratase activity and/or dehydroshikimate dehydratase activity is/arereduced as compared to an unmodified strain, particularly preferably amicroorganism which has been modified so that dehydroshikimatedehydratase activity is reduced as compared to an unmodified strain.

The term “having an ability to produce an organic acid” used hereinmeans that, when the microorganism is cultured in a medium, themicroorganism can produce and accumulate an organic acid in the medium.

The organic acid is not particularly restricted as long as it is anorganic acid which is not an aromatic carboxylic acid, and it ispreferably an amino acid or a carboxylic acid, more preferably acarboxylic acid, still more preferably an aliphatic carboxylic acid.Further, among these carboxylic acids, the organic acid is preferably apolycarboxylic acid, more preferably a dicarboxylic acid.

Examples of the organic acid which is not an aromatic carboxylic acidinclude lactic acid, succinic acid, malic acid, fumaric acid,oxaloacetic acid, citric acid, isocitric acid, 2-oxoglutaric acid,cis-aconitic acid, pyruvic acid, acetic acid and amino acids.Thereamong, the organic acid which is not an aromatic carboxylic acid ispreferably a dicarboxylic acid, more preferably succinic acid, malicacid, fumaric acid, citric acid, isocitric acid, 2-oxoglutaric acid,cis-aconitic acid or pyruvic acid, still more preferably succinic acid,malic acid or fumaric acid, particularly preferably succinic acid.

Further, the term “an ability to produce an aromatic carboxylic acid”used herein refers to an ability of the microorganism of the presentinvention to produce and accumulate an aromatic carboxylic acid in amedium when the microorganism is cultured in the medium. The expression“a reduced ability to produce an aromatic carboxylic acid” means thatthe ability to produce an aromatic carboxylic acid is reduced ascompared to an unmodified strain such as a wild-type strain. Here, theterm “unmodified strain” encompasses wild-type strains and those strainswhich have an ability to produce an aromatic carboxylic acid at a levelcomparable to a wild-type strain, as well as those strains having DAHPsynthase activity, dehydroquinate synthase activity, dehydroquinatedehydratase activity and dehydroshikimate dehydratase activity.

Examples of an aromatic carboxylic acid to be reduced during culturinginclude oxygen-containing heterocyclic aromatic carboxylic acids,nitrogen-containing heterocyclic aromatic carboxylic acids andbenzene-based aromatic carboxylic acids.

Examples of the oxygen-containing heterocyclic aromatic carboxylic acidsinclude those in which a carboxyl group is bound, directly or via alinking group, to an oxygen-containing heterocyclic ring having aromaticproperties, and specific examples thereof include furan monocarboxylicacids such as furoic acid and pyromucic acid; and furan dicarboxylicacids such dehydromucic acid.

Examples of the nitrogen-containing heterocyclic aromatic carboxylicacids include those in which a carboxyl group is bound, directly or viaa linking group, to a nitrogen-containing heterocyclic ring havingaromatic properties, and specific examples thereof include pyridinemonocarboxylic acids such as nicotinic acid, picolinic acid andisonicotinic acid; and hydroxypyridine carboxylic acids such ascitrazinic acid; pyridine dicarboxylic acids such as quinolinic acid,lutidinic acid, isocinchomeronic acid, dipicolinic acid, cinchomeronicacid, dinicotinic acid and uvitonic acid; and pyridine tricarboxylicacids such as berberonic acid.

Examples of the benzene-based aromatic carboxylic acids include those inwhich a carboxyl group is bound to a benzene ring directly or via alinking group, and the benzene ring has preferably a plurality ofsubstituents, more preferably three or more substituents. Thereamong,hydroxybenzene carboxylic acids having a hydroxyl group in addition to acarboxyl group are preferred and hydroxybenzene carboxylic acids havinga plurality of carboxyl groups are more preferred. Further,hydroxybenzene carboxylic acids which have a hydroxy group and aplurality of carboxyl groups at the same time are particularlypreferred. These hydroxybenzene carboxylic acids also includederivatives thereof which have a substituent in addition to a hydroxygroup and a carboxyl group.

More specific examples of the benzene-based aromatic carboxylic acidsinclude benzene monocarboxylic acids such as benzoic acid, toluic acid,xylic acid, α-toluic acid, cinnamic acid and hydrocinnamic acid; benzenedicarboxylic acids such as phthalic acid, isophthalic acid andterephthalic acid; benzene tricarboxylic acids such as hemimelliticacid, trimellitic acid and trimesic acid; hydroxybenzene carboxylicacids such as salicylic acid and creosotic acid; dihydroxybenzenecarboxylic acids such as pyrocatechuic acid, protocatechuic acid,resorcylic acid and gentisic acid; and trihydroxybenzene carboxylicacids such as gallic acid.

Thereamong, it is preferred that a furan dicarboxylic acid such asdehydromucic acid; a hydroxypyridine carboxylic acid such as citrazinicacid; a pyridine dicarboxylic acid such as quinolinic acid, lutidinicacid, isocinchomeronic acid, dipicolinic acid, cinchomeronic acid,dinicotinic acid or uvitonic acid; a pyridine tricarboxylic acid such asberberonic acid; a benzene dicarboxylic acid such as phthalic acid,isophthalic acid or terephthalic acid; a benzene tricarboxylic acid suchas hemimellitic acid, trimellitic acid or trimesic acid; ahydroxybenzene carboxylic acid such as salicylic acid or creosotic acid;a dihydroxybenzene carboxylic acid such as pyrocatechuic acid,protocatechuic acid, resorcylic acid or gentisic acid; or atrihydroxybenzene carboxylic acid such as gallic acid be reduced.

It is more preferred that a hydroxypyridine carboxylic acid such ascitrazinic acid; a pyridine dicarboxylic acid such as quinolinic acid,lutidinic acid, isocinchomeronic acid, dipicolinic acid, cinchomeronicacid, dinicotinic acid or uvitonic acid; a benzene dicarboxylic acidsuch as phthalic acid, isophthalic acid or terephthalic acid; a benzenetricarboxylic acid such as hemimellitic acid, trimellitic acid ortrimesic acid; a hydroxybenzene carboxylic acid such as salicylic acidor creosotic acid; or a dihydroxybenzene carboxylic acid such aspyrocatechuic acid, protocatechuic acid, resorcylic acid or gentisicacid be reduced.

It is still more preferred that a pyridine dicarboxylic acid such asquinolinic acid, lutidinic acid, isocinchomeronic acid, dipicolinicacid, cinchomeronic acid, dinicotinic acid or uvitonic acid; ahydroxybenzene carboxylic acid such as salicylic acid or creosotic acid;or a dihydroxybenzene carboxylic acid such as pyrocatechuic acid,protocatechuic acid, resorcylic acid or gentisic acid be reduced.

It is especially preferred that lutidinic acid, uvitonic acid,pyrocatechuic acid, protocatechuic acid, resorcylic acid or gentisicacid be reduced.

It is yet still more preferred that pyrocatechuic acid, protocatechuicacid, resorcylic acid or gentisic acid be reduced, and it isparticularly preferred that protocatechuic acid be reduced.

Although the details of the mechanism are not clear, causes ofcoloration are speculated to be that, during the later-described polymerpolymerization reaction, these aromatic carboxylic acids arecross-linked between the resulting polymers, or colored substances, inwhich these aromatic carboxylic acids are configured on a catalyst usedin the polymer synthesis, are generated. Therefore, it is preferred thatsuch coloration-causing aromatic carboxylic acid be reduced in theproduction of an organic acid since it allows the later-describedpurification process to be simplified and consequently, the productioncost of an organic acid can be reduced.

The concentration of an aromatic carboxylic acid in the later-describedculture medium or the resulting organic acid can be determined bymeasuring and analyzing the organic acid by conventionally known columnchromatography.

The microorganism according to the present invention may be amicroorganism which is obtained by modifying a microorganismintrinsically having an ability to produce an organic acid, or amicroorganism imparted with an ability to produce an organic acid bybreeding, so that the activity of at least one enzyme selected from thegroup consisting of DAHP synthase, dehydroquinate synthase,dehydroquinate dehydratase and dehydroshikimate dehydratase is reducedand the ability to produce an aromatic carboxylic acid is therebyreduced. Alternatively, the microorganism according to the presentinvention may also be a microorganism which has been modified as in theabove so that the ability to produce an aromatic carboxylic acid isreduced and then imparted with an ability to produce an organic acid.

Examples of a means for imparting an ability to produce an organic acidby breeding include mutation treatments and gene recombinationtreatments. For each organic acid, a known method, such as enhancementof the expression of the respective biosynthetic enzyme genes, can beemployed. For instance, when imparting an ability to produce succinicacid, a means for reducing the lactate dehydrogenase activity bymodification, a means for enhancing the pyruvate carboxylate activity orthe like may be employed.

The microorganism to be used in the present invention can be obtained byusing a microorganism shown below as a parent strain and modifying theparent strain. The type of the parent strain is not particularlyrestricted as long as it is a microorganism capable of producing anorganic acid and examples thereof include coryneform bacteria and thosebacteria belonging to the genera Mycobacterium, Rhodococcus, Nocardiaand Streptomyces; however, it is more preferably a coryneform bacterium.

The coryneform bacterium is not particularly restricted as long as it isclassified into coryneform bacteria, and examples thereof include thosebacteria belonging to the genera Corynebacterium, Brevibacterium andArthrobacter. Among these, those bacteria belonging to the generaCorynebacterium and Brevibacterium are preferred, and more preferredexamples include those bacteria classified as Corynebacteriumglutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes andBrevibacterium lactofermentum.

Particularly preferred specific examples of the parent strain of themicroorganism to be used in the present invention include Brevibacteriumflavum MJ-233 (FERM BP-1497), Brevibacterium flavum MJ-233 AB-41 (FERMBP-1498), Brevibacterium ammoniagenes ATCC6872, Corynebacteriumglutamicum ATCC31831 and Brevibacterium lactofermentum ATCC13869. It isnoted here that, since Brevibacterium flavum may currently be classifiedas Corynebacterium glutamicum (Lielbl, W., Ehrmann, M., Ludwig, W. andSchleifer, K. H., Int. J. Syst. Bacteriol., 1991, vol. 41, p 255-260),in the present invention, the Brevibacterium flavum MJ-233 strain andits mutant strain, MJ-233 AB-41 strain, are regarded as the same asCorynebacterium glutamicum MJ-233 strain and Corynebacterium glutamicumMJ-233 AB-41 strain, respectively.

Brevibacterium flavum MJ-233 has been deposited as of Apr. 28, 1975,with National Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry (currently, International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology; Tsukuba Central6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan) under the accessionNo. FERM P-3068 and converted to an international deposit under theaccession No. FERM BP-1497 as of May 1, 1981, under the Budapest Treaty.

Further, the above-described microorganisms to be used as a parentstrain may be not only a wild-type strain, but also any of mutantstrains obtained by a conventional mutation treatment such as UVirradiation or NTG treatment and recombinant strains induced by agenetic method such as cell fusion or genetic recombination.

In the following descriptions, the aromatic carboxylic acid is assumedto be protocatechuic acid.

Protocatechuic acid is synthesized from 3-dehydrosikimate via theshikimic acid pathway where a condensation reaction betweenphosphoenolpyruvate, which is an intermediate metabolite of theglycolytic pathway, and erythrose-4-phosphate, which is an intermediatemetabolite of the pentose phosphate pathway, is catalyzed by enzymessuch as DAHP synthase, dehydroquinate synthase, dehydroquinatedehydratase and dehydroshikimate dehydratase. The synthetic pathway ofprotocatechuic acid is shown in FIG. 1.

The microorganism to be used in the present invention can be obtained bymodifying the above-described strain so that at least one enzymeactivity selected from the group consisting of DAHP synthase activity,dehydroquinate synthase activity, dehydroquinate dehydratase activityand dehydroshikimate dehydratase activity is reduced as compared to anunmodified strain.

The term “DAHP synthase activity” refers to an activity to catalyze areaction in which phosphoenolpyruvate and erythrose-4-phosphate arecondensed to yield DAHP (3-deoxy-D-arabino-heptulosonate-7-phosphate)(EC: 2.5.1.54). The phrase “modified so that the DAHP synthase activityis reduced” means that the DAHP synthase activity is lower than that ofan unmodified strain such as wild-type strain. The DAHP synthaseactivity is reduced to preferably not higher than 30%, more preferablynot higher than 10% per unit cell weight, as compared to that of anunmodified strain. Further, the DAHP synthase activity may also becompletely lost. A reduction in the DAHP synthase activity can beverified by measuring the DAHP synthase activity in accordance with aknown method such as the one described in Liu, et al. (Liu Y. J., Li P.P., Zhao K. X., Wang B. J., Jiang C. Y., Drake H. L. and Liu S. J.,Appl. Environ. Microbiol., 2008, vol. 74(14), p 5497-5503).

The term “dehydroquinate synthase activity” refers to an activity tocatalyze a reaction in which 3-dehydroquinate is produced from DAHP(3-deoxy-D-arabino-heptulosonate-7-phosphate) (EC: 4.2.3.4). The phrase“modified so that the dehydroquinate synthase activity is reduced” meansthat the dehydroquinate synthase activity is lower than that of anunmodified strain such as wild-type strain. The dehydroquinate synthaseactivity is reduced to preferably not higher than 30%, more preferablynot higher than 10% per unit cell weight, as compared to that of anunmodified strain. Further, the dehydroquinate synthase activity mayalso be completely lost. A reduction in the dehydroquinate synthaseactivity can be verified by measuring the dehydroquinate synthaseactivity in accordance with a known method such as the one described inde Mendonca, et al. (de Mendonca J. D., Ely F., Palma M. S., Frazzon J.,Basso L. A. and Santos D. S., J. Bacteriol., 2007, vol. 189(17), p6246-6252).

The term “dehydroquinate dehydratase activity” refers to an activity tocatalyze a reaction in which 3-dehydroshikimate is produced from3-dehydroquinate (EC: 4.2.1.10). The phrase “modified so that thedehydroquinate dehydratase activity is reduced” means that thedehydroquinate dehydratase activity is lower than that of an unmodifiedstrain such as wild-type strain. The dehydroquinate synthase activity isreduced to preferably not higher than 30%, more preferably not higherthan 10% per unit cell weight, as compared to that of an unmodifiedstrain. Further, the dehydroquinate dehydratase activity may also becompletely lost. A reduction in the dehydroquinate dehydratase activitycan be verified by measuring the dehydroquinate dehydratase activity inaccordance with a known method such as the one described in Elsemore, etal. (Elsemore D. A. and Ornston L. N., J. Bacteriol., 1995, vol.177(20), p 5971-5978).

The term “dehydroshikimate dehydratase activity” refers to an activityto catalyze a reaction in which protocatechuic acid is produced from3-dehydroshikimate (EC: 4.2.1.-). The phrase “modified so that thedehydroshikimate dehydratase activity is reduced” means that thedehydroshikimate dehydratase activity is lower than that of anunmodified strain such as wild-type strain. The dehydroshikimatedehydratase activity is reduced to preferably not higher than 30%, morepreferably not higher than 10% per unit cell weight, as compared to thatof an unmodified strain. Further, the dehydroshikimate dehydrataseactivity may be completely lost as well. A reduction in thedehydroshikimate dehydratase activity can be verified by measuring thedehydroshikimate dehydratase activity in accordance with a known methodsuch as the one described in Elsemore, et al. (Elsemore D. A. andOrnston L. N., J. Bacteriol., 1995, vol. 177(20), p 5971-5978).

A strain which has been modified so that at least one enzyme activityselected from the group consisting of DAHP synthase activity,dehydroquinate synthase activity, dehydroquinate dehydratase activityand dehydroshikimate dehydratase activity is reduced as compared to anunmodified strain can be obtained by treating the above-described parentstrain with a mutagenic agent normally used in a mutation treatment,such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, andthen selecting a strain in which the enzyme activity of interest isreduced.

Alternatively, such a strain may also be obtained by modifying a parentstrain with the genes encoding the respective enzymes. Specifically,this can be achieved by, for example, disrupting a gene on a chromosomeor modifying a promoter or an expression control sequence such asSine-Dalgarno (SD) sequence.

A DAHP synthase gene on a chromosome is not particularly restricted aslong as it encodes a protein having DAHP synthase activity, and examplesthereof include genes originated from Corynebacterium glutamicum MJ-233strain which comprises the nucleotide sequence shown in SEQ ID NO:7 or 9(hereinafter, also referred to as “the aroF gene” and “the aroG gene”,respectively).

Further, a dehydroquinate synthase gene, a dehydroquinate dehydratasegene and a dehydroshikimate dehydratase gene are also not particularlyrestricted as long as they encode a protein having the respective enzymeactivity, and examples thereof include genes originated fromCorynebacterium glutamicum MJ-233 strain, each of which comprises thenucleotide sequence shown in SEQ ID NO:11, 13 or 15 (hereinafter, alsoreferred to as “the aroB gene”, “the qsuC gene” and “the qsuB gene”,respectively).

Further, the aroF gene, aroG gene, aroB gene, qsuC gene and qsuB genemay also each be a gene which encodes a protein comprising the aminoacid sequence as shown in SEQ ID NO:8, 10, 12, 14 or 16 except that oneor several amino acids are substituted, deleted, inserted or added, aslong as the resulting protein has the respective enzyme activity. Here,the term “several amino acids” usually means 2 or more, and not morethan 20 amino acids, preferably not more than 10 amino acids, morepreferably not more than 5 amino acids.

Further, in accordance with the type of the microorganism to be used asa host, it is also possible to use a DAHP synthase gene, adehydroquinate synthase gene, a dehydroquinate dehydratase gene and adehydroshikimate dehydratase gene that are originated from a bacteriumother than coryneform bacterium or other microorganism. As the DAHPsynthase gene, dehydroquinate synthase gene, dehydroquinate dehydratasegene and dehydroshikimate dehydratase gene that are originated from amicroorganism, for example, genes whose nucleotide sequences havealready been determined or genes encoding a protein having an activityof DAHP synthase, dehydroquinate synthase, dehydroquinate dehydratase ordehydroshikimate dehydratase, which are isolated from a chromosome of amicroorganism based on the homology or the like to determine thenucleotide sequences thereof, may be used. In addition, once thenucleotide sequences are determined, genes that are synthesized inaccordance with the thus determined nucleotide sequences may also beused. These genes can be obtained by amplifying a region containing therespective promoter thereof and an ORF region in accordance with ahybridization method or a PCR method.

A method of disrupting the qsuB gene in a coryneform bacterium will nowbe described. The qsuB gene can be acquired by, for example,synthesizing a synthetic oligonucleotide based on the above-describedsequence and then cloning the thus obtained oligonucleotide by PCR usinga chromosomal DNA of Corynebacterium glutamicum as a template. Achromosomal DNA can be prepared from a DNA donor bacterium by, forexample, the method of Saito and Miura (see Saito H. and Miura K.,Biochim Biophys Acta., 1963, vol. 72, p 619-629; Text for BioengineeringExperiments, edited by the Society for Biotechnology, Japan, p 97-98,Baifukan, 1992).

The qsuB gene prepared in the above-described manner or a part thereofcan be used for gene disruption. It is noted here that, since the geneto be disrupted may be any gene as long as it has a homology (identity)at such a level that induces homologous recombination with the qsuB geneon a chromosomal DNA of the bacterium to be disrupted, a gene having ahomology to the sequence shown in SEQ ID NO:15 can also be used. Here,the “homology at such a level that induces homologous recombination” ispreferably not less than 80%, more preferably not less than 90%,particularly preferably not less than 95%. Further, homologousrecombination may occur between any DNAs as long as they can hybridizewith the above-described gene (complementary strand of SEQ ID NO:15)under stringent conditions. Here, the stringent conditions may be, forexample, those where genes are hybridized under a washing condition ofconventional Southern hybridization comprising the salt concentrationsequivalent to 1×SSC and 0.1% SDS at 60° C., preferably a conditioncomprising the salt concentrations equivalent to 0.1×SSC and 0.1% SDS at60° C.

Further, as the aroF gene, aroG gene, aroB gene and qsuC gene, geneshaving a homology to the sequences shown in SEQ ID NO:7, 9, 11 and 13(DNAs which can hybridize with the complementary strands of SEQ ID NO:7,9, 11 and 13 under stringent conditions) can be used, respectively.

Using the above-described gene, the qsuB gene on a chromosome can bedisrupted by, for example, deleting a partial sequence of the qsuB geneto prepare a defective qsuB gene which has been modified not to producenormal QsuB protein; transforming a coryneform bacterium with a DNAcontaining the gene; and then allowing the gene on the chromosome toundergo recombination with the defective gene. Such gene disruption bygene substitution utilizing homologous recombination has already beenestablished and examples thereof include a method in which a linear DNAis used and a method in which a plasmid containing atemperature-sensitive replication origin is used (U.S. Pat. No.6,303,383 and JP H05-007491A). Further the above-described genedisruption by gene substitution utilizing homologous recombination canalso be performed by using a plasmid which does not have replicationcapacity in a host. As such a plasmid which does not have replicationcapacity in a coryneform bacterium, a plasmid having replicationcapacity in Escherichia coli is preferred, and examples thereof includepHSG299 (manufactured by Takara Bio Inc.) and pHSG399 (manufactured byTakara Bio Inc.). In the above, a case where the qsuB gene is disruptedin a coryneform bacterium was described; however, disruption of the aroFgene, aroG gene, aroB gene or qsuC gene as well as disruption in otherbacteria can also be achieved in the same manner as described above.

In order to obtain a microorganism having a reduced ability to producean aromatic carboxylic acid such as protocatechuic acid, as described inthe above, a microorganism modified so that the activity of at least oneenzyme selected from the group consisting of DAHP synthase,dehydroquinate synthase, dehydroquinate dehydratase and dehydroshikimatedehydratase is reduced may be employed; however, a microorganism whichhas been modified so that the dehydroshikimate dehydratase activity isreduced is preferably used. The reason therefor is as follows. When theactivity of DAHP synthase, dehydroquinate synthase or dehydroquinatedehydratase is reduced, not only the synthesis of protocatechuic acid,but also the synthesis of metabolites such as phenylalanine that aresynthesized by the shikimic acid pathway and required for growth becomedifficult, so that growth tends to be suppressed. In fact, it isreported that the growth of a coryneform bacterium having a disruptionin the gene encoding DAHP synthase was inhibited on a synthetic medium(Liu Y. J., Li P. P., Zhao K. X., Wang B. J., Jiang C. Y., Drake H. L.,Liu S. J., Appl. Environ. Microbiol., 2008, vol. 74(14), p 5497-5503).Accordingly, in cases where the activities of these enzymes are reduced,it is required to add an aromatic amino acid such as phenylalanine inorder to allow the microorganism to grow. Meanwhile, since the shikimicacid pathway is not blocked even when the dehydroshikimate dehydrataseactivity is reduced, it is believed that such deterioration in growthdoes not occur. Accordingly, a microorganism which has been modified sothat the activity of dehydroshikimate dehydratase is reduced ispreferred because the amount of aromatic amino acids such asphenylalanine or organic nitrogen containing an aromatic amino acid thatare required to be added for growth may be small and, therefore, such amicroorganism can be grown on a simple medium such as synthetic medium,which is economical.

In order to obtain a microorganism having a reduced ability to producean aromatic carboxylic acid such as protocatechuic acid, as described inthe above, either a microorganism which has been modified so that theactivity of at least one enzyme selected from the group consisting ofDAHP synthase, dehydroquinate synthase, dehydroquinate dehydratase anddehydroshikimate dehydratase is reduced or a microorganism which hasbeen modified so that the activity of shikimate dehydrogenase isenhanced may be employed.

The term “shikimate dehydrogenase activity” refers to an activity tocatalyze a reaction in which shikimate is produced from3-dehydroshikimate (EC: 1.1.1.25, 1.1.1.282, 1.1.5.8). The phrase“modified so that the shikimate dehydrogenase activity is enhanced”means that the shikimate dehydrogenase activity is higher than that ofan unmodified strain such as wild-type strain. The shikimatedehydrogenase activity is increased to preferably not less than 1.5times, more preferably not less than 3 times per unit cell weight, ascompared to that of an unmodified strain. An enhancement in theshikimate dehydrogenase activity can be verified by measuring theshikimate dehydrogenase activity in accordance with a known method suchas the one described in Fonseca, et al. (Fonseca I. O., Magalhaes M. L.,Oliveira J. S., Silva R. G., Mendes M. A., Palma M. S., Santos D. S. andBasso L. A., Protein Expr. Purif., 2006, vol. 46(2), p 429-437).

A strain in which the shikimate dehydrogenase activity is enhanced canbe obtained by treating a parent strain with a mutagenic agent normallyused in a mutation treatment, such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, and thenselecting a strain having an increased shikimate dehydrogenase activity.

Alternatively, such a strain may also be obtained by modifying a parentstrain with the gene encoding the shikimate dehydrogenase. Specifically,the shikimate dehydrogenase activity can be enhanced by increasing thecopy number of the shikimate dehydrogenase gene, and the copy number canbe increased by, for example, using a plasmid or increasing the numberof copies on a chromosome by a known homologous recombination method.Further, an enhancement of the shikimate dehydrogenase activity can alsobe achieved by introducing a mutation to a promoter of the shikimatedehydrogenase gene on a chromosome or a plasmid or by allowing theshikimate dehydrogenase gene to be highly expressed by substitution orthe like thereof into a stronger promoter.

The shikimate dehydrogenase gene is not particularly restricted as longas it encodes a protein having shikimate dehydrogenase activity, andexamples thereof include a gene originated from Corynebacteriumglutamicum MJ-233 strain which comprises the nucleotide sequence shownin SEQ ID NO:17 (hereinafter, also referred to as “the qsuD gene”).

Further, the qsuD gene may also be, as long as it encodes a proteinhaving shikimate dehydrogenase activity, a DNA which hybridizes with aDNA having a sequence complementary to the above-described nucleotidesequence under stringent conditions or a homolog such as a DNA which hasa homology of not less than 80%, preferably not less than 90%, morepreferably not less than 95%, particularly preferably not less than 99%to the above-described nucleotide sequence. Here, the stringentconditions may be, for example, those where genes are hybridized under awashing condition of conventional Southern hybridization comprising thesalt concentrations equivalent to 1×SSC and 0.1% SDS at 60° C.,preferably a condition comprising the salt concentrations equivalent to0.1×SSC and 0.1% SDS at 60° C.

Further, the qsuD gene may also be a gene which encodes a proteincomprising the amino acid sequence as shown in SEQ ID NO:18 except thatone or several amino acids are substituted, deleted, inserted or added,as long as the resulting protein has the shikimate dehydrogenaseactivity. Here, the term “several amino acids” usually means 2 or more,but not more than 20 amino acids, preferably not more than 10 aminoacids, more preferably not more than 5 amino acids.

Further, a shikimate dehydrogenase gene originated from a non-coryneformbacterium, other microorganism, an animal or a plant may also be used.As the shikimate dehydrogenase gene originated from other microorganism,an animal or a plant, for example, a gene whose nucleotide sequence hasalready been determined or a gene encoding a protein having shikimatedehydrogenase activity which is isolated from a chromosome of amicroorganism, an animal or a plant based on the homology or the like todetermine the nucleotide sequence thereof, may be used. In addition,once the nucleotide sequence is determined, a gene synthesized inaccordance with the thus determined nucleotide sequence may also beused. These genes can be obtained by amplifying a region containing therespective promoter thereof and an ORF region in accordance with ahybridization method or a PCR method.

By inserting the thus isolated gene encoding shikimate dehydrogenaseinto a known expression vector so that the gene can be expressed, ashikimate dehydrogenase expression vector is provided. By performingtransformation with this expression vector, a shikimate dehydrogenaseactivity-enhanced strain can be obtained. Alternatively, a shikimatedehydrogenase activity-enhanced strain can also be obtained byincorporating a DNA encoding shikimate dehydrogenase into a chromosomalDNA of a host bacterium by homologous recombination or the like so thatthe incorporated DNA can be expressed. These transformation andhomologous recombination can be carried out in accordance with aconventional method known to those skilled in the art.

When introducing a shikimate dehydrogenase gene into a chromosome or aplasmid, an appropriate promoter and more preferably a terminator areincorporated into a 5′-upstream region and a 3′-downstream region of thegene, respectively. These promoter and terminator are not particularlyrestricted as long as they are known to function in a bacterium to beused as a host, and they may be the promoter and terminator of theshikimate dehydrogenase gene itself or may be replaced with otherpromoter and terminator. The vectors, promoters, terminators and thelike that can be used in a variety of bacteria are described in detailin, for example, “Fundamental Microbiology (Biseibutsugaku Kiso-kouza)8; Genetic Engineering, Kyoritsu Shuppan Co., Ltd.”.

A method of enhancing the qsuD gene in a coryneform bacterium will nowbe described. In cases where a coryneform bacterium is used, arecombinant plasmid capable of enhancing the expression of the qsuD genein the coryneform bacterium can be obtained by introducing a DNAfragment containing the qsuD gene into a plasmid vector which contains agene regulating the replication and growth functions of a plasmid in thecoryneform bacterium. By transforming a coryneform bacterium such asCorynebacterium glutamicum MJ-233 strain with the thus obtainedrecombinant vector, a coryneform bacterium having an enhanced expressionof the qsuD gene can be obtained. This transformation can be carried outby, for example, an electric pulse method (Vertes A. A., Inui M.,Kobayashi M., Kurusu Y. and Yukawa H., Res. Microbiol., 1993, vol.144(3), p 181-185).

A plasmid vector capable of introducing a gene into a coryneformbacterium is not particularly restricted as long as it contains at leasta gene which regulates the replication and growth functions in thecoryneform bacterium. Specific examples of such plasmid vector includethe plasmid pCRY30 described in JP H3-210184A; the plasmids pCRY21,pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX, which are described in JPH2-72876A and U.S. Pat. No. 5,185,262; the plasmids pCRY2 and pCRY3which are described in JP H1-191686A; the plasmid pAM330 described in JPS58-67679A; the plasmid pHM1519 described in JP S58-77895A; the plasmidspAJ655, pAJ611 and pAJ1844, which are described in JP S58-192900A; theplasmid pCG1 described in JP S57-134500A; the plasmid pCG2 described inJP S58-35197A; and the plasmids pCG4 and pCG11 which are described in JPS57-183799A. Thereamong, as a plasmid vector to be used in a host-vectorsystem of a coryneform bacterium, a plasmid vector which contains a generegulating the replication and growth functions of a plasmid in thecoryneform bacterium and a gene regulating the function of stabilizingthe plasmid in the coryneform bacterium is preferred. For example, theplasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KXcan be suitably employed.

In the above-described recombinant plasmid or incorporation into achromosome, a promoter used to express the qsuD gene may be any promoteras long as it functions in the coryneform bacterium, and it may also bethe promoter of the qsuD gene itself. The expression level of the qsuDgene can also be adjusted by appropriately selecting the promoter. Inthe above, a case where a coryneform bacterium is used was described;however, enhancement of the shikimate dehydrogenase activity in otherbacteria can also be achieved in the same manner as described above.

The microorganism according to the present invention may also be abacterium which is, in addition to being modified so that the ability toproduce an aromatic carboxylic acid is reduced (modification by whichthe activity of at least one enzyme selected from the group consistingof DAHP synthase, dehydroquinate synthase, dehydroquinate dehydrataseand dehydroshikimate dehydratase is reduced or the shikimatedehydrogenase activity is enhanced), modified so that the lactatedehydrogenase (hereinafter, also referred to as “LDH”) activity isreduced. The term “LDH activity” used herein refers to an activity tocatalyze a reaction in which pyruvate is reduced to yield lactate (EC:1.1.1.27). The phrase “the LDH activity is reduced” means that the LDHactivity is lower than that of an unmodified strain. The LDH activity isreduced to preferably not higher than 30%, more preferably not higherthan 10% per unit cell weight, as compared to that of an unmodifiedstrain. Further, the LDH activity may also be completely lost. Areduction in the LDH activity can be verified by measuring the LDHactivity in accordance with a known method such as the one described inKanarek, et al. (Kanarek L. and Hill R. L., J. Biol. Chem., 1964, vol.239, p 4202-4206).

Specific examples of a method for preparing a strain in which the LDHactivity is reduced include the method described in JP H11-206385A whichutilizes homologous recombination on a chromosome and the method inwhich the sacB gene is used (Schafer A., Tauch A., Jager W., KalinowskiJ., Thierbach G. and Puhler A., Gene, 1994, vol. 145(1), p 69-73). Abacterium in which the LDH activity and the ability to produce anaromatic carboxylic acid are both reduced can be obtained by, forexample, preparing a bacterium whose ldh gene is disrupted and thenmodifying the bacterium so that the ability to produce an aromaticcarboxylic acid is reduced. It is noted here, however, that whicheverthis modification operation for reducing the LDH activity or themodification operation for reducing the ability to produce an aromaticcarboxylic acid may be performed first.

Further, the microorganism to be used in the present invention may alsobe a bacterium which is, in addition to being modified so that theability to produce an aromatic carboxylic acid is reduced (modificationby which the activity of at least one enzyme selected from the groupconsisting of DAHP synthase, dehydroquinate synthase, dehydroquinatedehydratase and dehydroshikimate dehydratase is reduced or the shikimatedehydrogenase activity is enhanced), modified so that the pyruvatecarboxylase (hereinafter, also referred to as “PC”) activity isenhanced. The term “PC activity” used herein refers to an activity tocatalyze a reaction in which pyruvate is carboxylated to yieldoxaloacetate (EC: 6.4.1.1). The phrase “the PC activity is enhanced”means that the PC activity is higher than that of an unmodified strain.The PC activity is increased to preferably not less than 1.5 times, morepreferably not less than 3 times per unit bacterial weight, as comparedto that of an unmodified strain. An enhancement in the PC activity canbe verified by measuring the PC activity in accordance with a knownmethod such as the one described in Fisher, et al. (Fisher S. H. andMagasanik B., J. Bacteriol., 1984, vol. 158(1), p 55-62).

A strain in which the PC activity is enhanced can be prepared in thesame manner as the above-described method of enhancing the shikimatedehydrogenase activity. More specifically, such a strain can be preparedby, for example, in the same manner as the method described in JPH11-196888A, introducing a pc gene into a coryneform bacterium to allowthe gene to be highly expressed. As a specific pc gene, for example, thepc gene originated from Corynebacterium glutamicum (Peters-Wendisch P.G., Kreutzer C., Kalinowski J., Patek M., Sahm H. and Eikmanns B. J.,Microbiology, 1998, vol. 144, p 915-927) may be used. Further, as the pcgene, a DNA which hybridizes with the pc gene originated fromCorynebacterium glutamicum under stringent conditions or a DNA encodinga protein having PC activity which has a homology of not less than 80%,preferably not less than 90%, more preferably not less than 95%,particularly preferably not less than 99% to the nucleotide sequence ofthe pc gene can also be suitably used.

Moreover, a pc gene originated from a coryneform bacterium other thanCorynebacterium glutamicum or other microorganism, or ananimal/plant-derived pc gene can also be used. Particularly, thesequences of the pc genes originated from the following microorganisms,plants or animals are already known (references are provided below) andthese pc genes can be obtained by performing hybridization in the samemanner as described in the above or by amplifying the ORF region thereofby PCR.

-   Human [Biochem. Biophys. Res. Comm., 202, 1009-1014, (1994)]-   Mouse [Proc. Natl. Acad. Sci. USA., 90, 1766-1779, (1993)]-   Rat [GENE, 165, 331-332, (1995)]-   Yeast; Saccharomyces cerevisiae [Mol. Gen. Genet., 229, 307-315,    (1991)]-   Schizosaccharomyces pombe [DDBJ Accession No. D78170]-   Bacillus stearothermophilus [GENE, 191, 47-50, (1997)]-   Rhizobium etli [J. Bacteriol., 178, 5960-5970, (1996)]

A bacterium in which the PC activity is enhanced and the ability toproduce an aromatic carboxylic acid is reduced can be obtained by, forexample, preparing a bacterium in which a pc gene is introduced andhighly expressed and then modifying the bacterium so that the ability toproduce an aromatic carboxylic acid is reduced. It is noted here,however, that whichever this modification operation for enhancing the PCactivity or the modification operation for reducing the ability toproduce an aromatic carboxylic acid may be performed first.

Further, the microorganism according to the present invention may alsobe a bacterium which is, in addition to being modified so that theability to produce an aromatic carboxylic acid is reduced (modificationby which the activity of at least one enzyme selected from the groupconsisting of DAHP synthase, dehydroquinate synthase, dehydroquinatedehydratase and dehydroshikimate dehydratase is reduced or the shikimatedehydrogenase activity is enhanced), modified so that the activity of atleast one enzyme selected from the group consisting of acetate kinase(hereinafter, also referred to as “ACK”) and phosphotransacetylase(hereinafter, also referred to as “PTA”) is reduced.

The term “PTA activity” refers to an activity to catalyze a reaction inwhich phosphate is transferred to acetyl-CoA to yield acetyl phosphate(EC: 2.3.1.8). The phrase “modified so that the PTA activity is reduced”means that the PTA activity is lower than that of an unmodified strain.The PTA activity is reduced to preferably not higher than 30%, morepreferably not higher than 10% per unit cell weight, as compared to thatof an unmodified strain. Further, the PTA activity may also becompletely lost. A reduction in the PTA activity can be verified bymeasuring the PTA activity in accordance with a known method such as theone described in Klotzsch (Klotzsch H. R., Meth. Enzymol., 1969, vol.12, p 381-386).

The term “ACK activity” refers to an activity to catalyze a reaction inwhich acetic acid is produced from acetyl phosphate and ADP (EC:2.7.2.1). The phrase “modified so that the ACK activity is reduced”means that the ACK activity is lower than that of an unmodified strain.The ACK activity is reduced to preferably not higher than 30%, morepreferably not higher than 10% per unit cell weight, as compared to thatof an unmodified strain. Further, the ACK activity may be completelylost as well. A reduction in the ACK activity can be verified bymeasuring the ACK activity in accordance with a known method such as theone described in Ramponi (Ramponi G., Meth. Enzymol., 1975, vol. 42, p409-426).

The activity of only either one of PTA and ACK may be reduced; however,in order to efficiently reduce the by-production of acetic acid, it ismore preferred that the activities of both enzymes be reduced.

It is noted here that, in Corynebacterium glutamicum (including thosebacteria classified as Brevibacterium flavum), as described inMicrobiology. 1999 February; 145(Pt 2):503-13, since both enzymes of PTAand ACK are encoded by the pta-ack operon (GenBank Accession No.X89084), the activities of both enzymes can be reduced by disrupting thepta gene.

A strain in which the activities of PTA and ACK are reduced can beobtained by disrupting the genes of these enzymes in accordance with aknown method such as a method which utilizes homologous recombination orthe method in which the sacB gene is used (Schafer A., Tauch A., JagerW., Kalinowski J., Thierbach G. and Puhler A., Gene, 1994, vol. 145(1),p 69-73). Specifically, such a strain can be obtained by the methoddisclosed in JP 2006-000091A. As the pta gene and the ack gene, inaddition the above-described gene having the nucleotide sequence ofGenBank Accession No. X89084, a gene having a homology at such a levelthat induces homologous recombination with the pta gene and the ack geneon the host chromosome can also be used. Here, the “homology at such alevel that induces homologous recombination” is preferably not less than80%, more preferably not less than 90%, particularly preferably not lessthan 95%. Further, homologous recombination may occur between any DNAsas long as they can hybridize with the above-described genes understringent conditions.

A bacterium in which the activity of at least one enzyme selected fromthe group consisting of PTA and ACK as well as the ability to produce anaromatic carboxylic acid are both reduced can be obtained by, forexample, preparing a bacterium whose pta gene and ack gene are disruptedand then modifying the bacterium so that the ability to produce anaromatic carboxylic acid is reduced. It is noted here, however, thatwhichever this modification operation for reducing the activities of PTAand ACK or the modification operation for reducing the ability toproduce an aromatic carboxylic acid may be performed first.

Further, the microorganism used in the present invention may also be abacterium which is obtained by performing two or more of theabove-described modifications in combination with the modification forreducing the ability to produce an aromatic carboxylic acid(modification by which the activity of at least one enzyme selected fromthe group consisting of DAHP synthase, dehydroquinate synthase,dehydroquinate dehydratase and dehydroshikimate dehydratase is reducedor the shikimate dehydrogenase activity is enhanced). When pluralmodifications are performed, their order is not particularly restricted.

<Method of Producing Organic Acid>

The method of producing an organic acid according to the presentinvention comprises the step of allowing the above-describedmicroorganism or a treated cell thereof to act on an organic rawmaterial to produce an organic acid. Particularly, it is preferred thatan organic acid be produced by allowing the above-describedmicroorganism or a treated cell thereof to act on an organic rawmaterial and then the resulting organic material be recovered.

Examples of the types of organic acids that can be produced and examplesof preferred organic acids are as described in the above.

In cases where the above-described microorganism is used in theproduction of an organic acid, the microorganism may be cultured on asolid slant medium such as an agar medium and then directly used for thereaction; however, it is preferred that the microorganism be cultured inadvance in a liquid medium (seed culture) and then used. The medium usedfor the seed culture may be any conventional medium used for culturing amicroorganism. For example, a common culture medium which is prepared byadding natural nutrient sources, such as meat extract, yeast extract andpeptone, to a composition composed of inorganic salts such as ammoniumsulfate, potassium phosphate and magnesium sulfate can be employed.After the seed culture, it is preferred that the resulting bacterialcells be recovered by centrifugation, membrane separation or the likeand then used for the reaction to produce an organic acid. An organicacid may be produced by allowing the seed-cultured microorganism toreact with an organic raw material while allowing the microorganism togrow in a medium containing the organic raw material. Alternatively, anorganic acid may also be produced by allowing the bacterial cellsobtained by culturing in advance to react with an organic raw materialin a reaction solution containing the organic raw material.

In the present invention, it is also possible to use a treated cell of amicroorganism. Examples of the treated cell include bacterial cellsimmobilized with acrylamide, carrageenan or the like; a homogenateprepared by pulverizing bacterial cells; a centrifugation supernatantthereof; and a fraction obtained by partially purifying the supernatantwith ammonium sulfate or the like.

The organic raw material to be used in the production method accordingto the present invention is not particularly restricted as long as it isa carbon source which can be assimilated by the microorganism of thepresent invention to produce succinic acid. As the organic raw material,usually, a fermentable sugar, for example, a carbohydrate such asgalactose, lactose, glucose, fructose, glycerol, sucrose, saccharose,starch or cellulose or a polyalcohol such as glycerin, mannitol, xylitolor ribitol is used. Thereamong, glucose, sucrose or fructose ispreferably used, and glucose or sucrose is particularly preferably used.

Further, a saccharified starch solution, molasses or the like, whichcontains the above-described fermentable sugar, may also be used, andspecifically, a sugar solution collected from a plant such as sugarcane,sugar beet or sugar maple is preferred.

These sugars may be used individually or in combination. Theconcentration at which the above-described sugar is used is notparticularly restricted; however, it is advantageous to increase theconcentration as much as possible within the range which does notinhibit the production of succinic acid. The concentration of theabove-described sugar is, with respect to the reaction solution, usuallynot lower than 5% (W/V), preferably not lower than 10% (W/V), and onanother front, usually not higher than 30% (W/V), preferably not higherthan 20% (W/V). Further, the above-described sugar may also be furtheradded in response to a decrease thereof associated with the progressionof the reaction.

The reaction solution containing the above-described organic rawmaterial is not particularly restricted and it may be, for example, amedium for culturing a microorganism or a buffer solution such asphosphate buffer. The reaction solution is preferably an aqueoussolution containing a nitrogen source, an inorganic salt and the like.Here, the nitrogen source is not particularly restricted as long as itcan be assimilated by the microorganism of the present invention toproduce succinic acid, and specific examples of such nitrogen sourceinclude a variety of organic and inorganic nitrogen compounds such asammonium salts, nitrates, urea, soybean hydrolysates, casein digests,peptone, yeast extracts, meat extracts and corn steep liquors. As theinorganic salt, a variety of phosphates, sulfates and metal salts ofmagnesium, potassium, manganese, iron, zinc and the like may be used.Further, a growth-promoting factor(s) such as vitamins (e.g., biotin,pantothenic acid, inositol and nicotinic acid), nucleotides and aminoacids may be added as required. Moreover, in order to suppress foamformation during reaction, it is desired to add an appropriate amount ofa commercially available antifoaming agent to the reaction solution.

The reaction solution also contains a carbonate ion, bicarbonate ion orcarbon dioxide gas in addition to the above-described organic rawmaterial, nitrogen source and inorganic salt. The carbonate ion or thebicarbonate ion is supplied from magnesium carbonate, sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate or thelike, which can also be used as a neutralizing agent; however, asrequired, the ion may also be supplied from carbonic acid or bicarbonicacid, or a salt thereof, or carbon dioxide gas. Specific examples of thesalt of carbonic acid or bicarbonic acid include magnesium carbonate,ammonium carbonate, sodium carbonate, potassium carbonate, ammoniumbicarbonate, sodium bicarbonate and potassium bicarbonate. Further, thecarbonate ion or bicarbonate ion is added at a concentration of usuallynot lower than 1 mM, preferably not lower than 2 mM, more preferably notlower than 3 mM, and on another front, usually not higher than 500 mM,preferably not higher than 300 mM, more preferably not higher than 200mM. In cases where carbon dioxide gas is added, it is contained in anamount of usually not less than 50 mg, preferably not less than 100 mg,more preferably not less than 150 mg, and on another front, usually notmore than 25 g, preferably not more than 15 g, more preferably not morethan 10 g, per 1 L of the solution.

The pH of the reaction solution can be adjusted by adding thereto, forexample, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium hydroxide, calciumhydroxide, magnesium hydroxide or ammonia. The pH of the reaction isusually not lower than 5, preferably not lower than 5.5, more preferablynot lower than 6, and on another front, not higher than 10, preferablynot higher than 9.5, more preferably not higher than 9.0. Thus, duringthe reaction as well, the pH of the reaction solution is adjusted asrequired within the above-described range by adding thereto an alkalinesubstance, carbonate, urea or the like.

The optimum growth temperature of the microorganism to be used in thepresent reaction is not particularly restricted as long as it is atemperature at which the microorganism grows optimally; however, it isusually not lower than 25° C. but usually not higher than 35° C.,preferably not higher than 32° C., particularly preferably not higherthan 30° C. The term “optimum growth temperature” refers to atemperature at which the fastest growth rate is attained under theconditions used for the production of succinic acid.

Further, as a method of preparing bacterial cells more suitable for theproduction of succinic acid, the method described in JP 2008-259451A, inwhich culturing is performed by alternately repeating depletion andreplenishment of a carbon source at short intervals, can also beemployed.

The amount of the microorganism to be used in the reaction is notparticularly restricted; however, in terms of the wet cell weight, themicroorganism is used in an amount of usually not less than 1 g/L,preferably not less than 10 g/L, more preferably not less than 20 g/L,and on another front, usually not more than 700 g/L, preferably not morethan 500 g/L, more preferably not more than 400 g/L. The reaction timeis not particularly restricted; however, it is usually not short than 1hour, preferably not shorter than 3 hours, and usually not longer than168 hours, preferably not longer than 72 hours.

During the seed culturing of a microorganism, it is required that oxygenbe supplied by aeration and stirring. On the other hand, although thereaction for producing an organic acid such as succinic acid may becarried out with aeration and stirring, it may also be carried out underan anaerobic atmosphere where aeration is not performed and oxygen isthus not supplied. Here, the condition “under an anaerobic atmosphere”can be attained by, for example, a method in which the reaction iscarried out in a closed container with no aeration, a method in whichthe reaction is carried out with supply of an inert gas such as nitrogengas or a method in which the reaction is carried out while aerating thesystem with a carbon dioxide-containing inert gas.

By such a microbial reaction as described in the above, an organic acidsuch as succinic acid, fumaric acid, malic acid or pyruvic acid isproduced and accumulated in the reaction solution.

The amount of generated protocatechuic acid is, with respect to theamount of succinic acid produced in the culture medium after thereaction performed by allowing the microorganism of the presentinvention or a treated cell thereof to act on an organic raw material,usually not more than 500 ppm, preferably not more than 300 ppm, morepreferably not more than 150 ppm, still more preferably not more than 75ppm, particularly preferably not more than 50 ppm. There is noparticular restriction with regard to the lower limit.

Further, the amount of generated uvitonic acid is, with respect to theamount of succinic acid produced in the culture medium after thereaction performed by allowing the microorganism of the presentinvention or a treated cell thereof to act on an organic raw material,usually not more than 500 ppm, preferably not more than 300 ppm, morepreferably not more than 150 ppm, still more preferably not more than 75ppm, particularly preferably not more than 50 ppm. There is noparticular restriction with regard to the lower limit.

It is noted here that, also in cases where an organic acid other thansuccinic acid is produced, the concentration of protocatechuic acid andthat of uvitonic acid are each, with respect to the organic acid ofinterest in the culture medium after the reaction, usually not more than500 ppm, preferably not more than 300 ppm, more preferably not more than150 ppm, still more preferably not more than 75 ppm, particularlypreferably not more than 50 ppm.

The organic acid accumulated in the reaction solution (culture medium)can be recovered therefrom in accordance with a conventional method. Themethod of recovering from an organic acid from the reaction solution isnot particularly restricted as long as a composition containing thedesired organic acid can be obtained. By removing the microorganism,water and/or impurities from the reaction solution, an organic acid canbe recovered.

The method of removing the microorganism, water and/or impurities is notparticularly restricted and the removal can be achieved by a knownmethod such as concentration, extraction, crystallization, an activatedcarbon treatment, a hydrogenation treatment or an ion-exchange columntreatment or by an arbitrary combination of these known methods.

In the method of producing an organic acid according to the presentinvention, since the production of an aromatic carboxylic acid which isconventionally difficult to be removed by a crystallization treatment isreduced, it is preferred that the process of recovering an organic acidincludes a crystallization treatment.

One example of such method of recovering an organic acid which includescrystallization is a method in which a microorganism is removed from areaction solution and then, after concentrating the resulting reactionsolution and extracting an organic acid as required, the resultant issubjected to crystallization, solid-liquid separation and drying. Afterthe solid-liquid separation, for example, an activated carbon treatment,a hydrogenation treatment and/or an ion-exchange column treatment mayalso be performed.

A method of obtaining an organic acid, succinic acid, will now bedescribed. However, the method of obtaining succinic acid is notrestricted to the following description.

<Concentration>

In the present invention, a fermentation liquid obtained after themicrobial reaction may also be concentrated as appropriate inconsideration of the operability and efficiency in the subsequentpurification step. The concentration method is not particularlyrestricted and examples thereof include a method in which an inert gasis allowed to circulate; a method in which water is distilled away byheating; a method in which water is distilled away under reducedpressure; and a combination of these methods. Further, the concentrationoperation may be performed in either a batch system or a continuoussystem.

<Removal of Microorganism>

In cases where a fermentation liquid is used in the method of thepresent invention, it is preferred that the fermentation liquid be oneobtained after the removal of microorganism. The method of removing amicroorganism is not particularly restricted and, for example,sedimentation separation, centrifugation, filtration separation and amethod in which these techniques are combined can be employed.Industrially, the removal of microorganism is carried out by a methodsuch as centrifugation or a membrane-filtration separation. As for thecentrifugation, for example, centrifugal sedimentation or centrifugalfiltration can be used. In the centrifugation, the operating conditionsthereof are not particularly restricted; however, the separation isusually performed at a centrifugal force of 100 G to 100,000 G. Further,the operation may be performed in either a batch system or a continuoussystem.

Further, in the membrane-filtration separation, for example,microfiltration and/or ultrafiltration can be employed. The material ofthe membrane is not particularly restricted and, for example, an organicfilm of polyolefin, polysulfin, polyacrylonitrile, polyvinylidenefluoride or the like, or a membrane made of an inorganic material suchas ceramic can be used. Further, the operation method may either be adead-end type or a cross-flow type. In the membrane-filtrationseparation, since a microorganism often causes clogging of the membrane,for example, a method in which membrane filtration is performed afterroughly removing the microorganism by centrifugation or the like may beemployed as well.

In the production method according to the present invention, in caseswhere a neutralizing agent is used in the microbial reaction asdescribed in the above, a salt of succinic acid is obtained, which isthen converted into the desired succinic acid. This conversion intosuccinic acid may also be performed by a reactive crystallization methodwhich utilizes a weakly acidic organic acid having an acid dissociationconstant (pKa) higher than that of the desired succinic acid. Examplesof such organic acid include acetic acid.

Further, alternatively, an inorganic acid may be used to convert thesalt of succinic acid obtained in the above-described manner intosuccinic acid. Examples of the inorganic acid to be used in this methodinclude sulfuric acid, hydrochloric acid, carbonic acid, phosphoric acidand nitric acid. More specifically, in the case of succinic acidfermentation, when fermentation is carried out while neutralizing theproduced succinic acid with ammonia or magnesium hydroxide, ammoniumsuccinate or magnesium succinate is produced in the resultingfermentation liquid. By treating such a fermentation liquid containingammonium succinate or magnesium succinate with sulfuric acid or thelike, a succinic acid-containing aqueous solution can be obtained.

In the present invention, the term “succinic acid-containing solution”or “succinic acid-containing aqueous solution” refers to a solution oran aqueous solution which mainly contains succinic acid derived from abiomass resource. Accordingly, a solution or an aqueous solution whichmainly contains the above-described salt of succinic acid, such asammonium succinate or magnesium succinate, is indicated as“succinate-containing solution” or “succinate-containing aqueoussolution”. The term “mainly contain” used herein refers to a conditionin which the solution or the aqueous solution contains the subjectcomponent in an amount of usually not less than 50% by weight,preferably not less than 60% by weight, more preferably not less than70% by weight, particularly preferably not less than 90% by weight, withrespect to the total weight of all components except the solvent.

<Extraction>

In the present invention, from an aqueous solution which containssuccinic acid obtained by conversion with the above-described inorganicacid, succinic acid may be extracted by using, but not particularlylimited to, an organic solvent.

The organic solvent to be used in this method is usually an organicsolvent which has an inorganic value/organic value ratio (I/O value) of0.2 to 2.3 and a boiling point of not lower than 40° C. at normalpressure, more preferably an organic solvent which has an I/O value of0.3 to 2.0 and a boiling point of not lower than 40° C. at normalpressure, still more preferably an organic solvent which has an I/Ovalue of 0.3 to 2.0 and a boiling point of not lower than 60° C. atnormal pressure. By using such an organic solvent, succinic acid can beselectively extracted and efficiently separated from sugars and aminoacids. Further, by using an organic solvent having a boiling point ofnot lower than 40° C. at normal pressure, it becomes possible to avoidthe risk of the solvent being vaporized and ignited and a problem of adecrease in the efficiency of succinic acid extraction due tovaporization of the solvent as well as a problem of difficulty torecycle the solvent.

The inorganic value and the organic value are proposed by OrganicConceptual Diagram (“Systematic Organic Qualitative Analysis”, FujitaA., Kazamashobo Co., Ltd. (1974)). The ratio of the inorganic value andthe organic value is determined by calculating the respective valuesbased on the numerical values predetermined for the functional groupsconstituting the organic compound of interest.

Examples of the organic solvent which has an I/O value of 0.2 to 2.3 anda boiling point of not lower than 40° C. at normal pressure includeketone-based solvents such as methyl ethyl ketone, methyl isobutylketone and acetone; ether-based solvents such as tetrahydrofuran anddioxane; ester-based solvents such as ethyl acetate; nitrile-basedsolvents such as acetonitrile; and alcohols having a carbon chain of 3or more carbon atoms, such as propanol, butanol and octanol.

The I/O value and boiling point of each solvent are shown below.

I O I/O Boiling point Tetrahydrofuran 30 80 0.375 66.0 Methyl ethylketone 65 60 1.083 79.6 Methyl isobutyl ketone 65 120 0.542 94.2 Acetone65 40 1.625 56.1 Acetonitrile 70 40 1.750 81.1 Ethyl acetate 85 80 1.06377.2 Propanol 100 60 1.667 97.2 Isobutanol 100 70 1.429 108.0 Octanol100 160 0.625 179.8 Dioxane 40 80 0.500 101.3

In the extraction step, the organic solvent is added in a volume of,with respect to 1 volume of the succinic acid-containing aqueoussolution, usually not less than 0.5, preferably not less than 1 andusually not more than 5, preferably not more than 3.

The temperature of the extraction step may be any temperature as long asit is a temperature at which succinic acid can be extracted; however, itis usually not lower than 10° C., preferably not lower than 20° C.,while normally not higher than 90° C., preferably not higher than 85° C.

By the extraction step, succinic acid is recovered in the organicsolvent and impurities such as sugars, elemental nitrogen originatedfrom fermentation, ammonia originated from the fermentative bacterium,sulfur-containing impurities and metal cations are separated to someextent. Here, in order to extract succinic acid more efficiently, theextraction treatment with an organic solvent may be repeated a pluralityof time, or countercurrent extraction may be performed.

<Removal of Impurities>

It is important that, in addition to the elemental nitrogen contained inthe biomass resource, the amount of many impurities such as elementalnitrogen and ammonia originated from the fermentative bacterium,sulfur-containing impurities and metal cations be reduced from thesuccinic acid-containing fermentation liquid by purification. Further,it is also important to reduce the amount of impurities showingabsorption in the ultraviolet region of 250 to 300 nm, particularly theamount of impurities containing an aromatic carboxylic acid, to a levelwhere an average absorbance of 0.05 or lower is attained (see JP2010-100617A).

In order to reduce the impurities contained in succinic acid that showabsorbance in the ultraviolet region of 250 to 300 nm, particularlyaromatic dicarboxylic acids, to a level where the average absorbancebecomes 0.05 or lower, it is usually required to subject the succinicacid produced in the above-described manner to a combination ofpurification treatments such as a crystallization treatment, anactivated carbon treatment, a hydrogenation treatment and a dryingtreatment. However, since the succinic acid-containing solution obtainedby the fermentation method (organic acid production method) according tothe present invention contains a markedly small amount of theabove-described impurities as compared to a solution obtained by aconventional fermentation method (organic acid production method), thepurification step of a crystallization treatment, an activated carbontreatment and the like can be reduced and the conditions thereof can berelaxed, so that the cost of producing an organic acid such as succinicacid can be reduced and the yield thereof can be improved, which arepreferred.

<Crystallization>

In the present invent, succinic acid may also be recovered bycrystallization from the succinic acid-containing solution obtained bythe reaction. By performing a crystallization operation, the amount ofaromatic carboxylic acids such as protocatechuic acid can be furtherreduced. In addition, in cases where crystallization is performed, ascompared to a case where a conventional microorganism is employed, thenumber of crystallization operations can be reduced, which is useful.

In the present invention, the term “succinic acid crystallization”refers to an operation in which crystals of succinic acid are formedfrom a succinic acid-containing solution by changing the solubility ofsuccinic acid with an application of a modification of some sort to thesuccinic acid-containing solution. For the succinic acidcrystallization, any method may be employed as long as it is anoperation which allows crystals of succinic acid to be formed from asuccinic acid-containing fluid. More specifically, examples of suchmethod include a cooling crystallization method in which succinic acidis precipitated by changing the temperature of a succinicacid-containing solution to utilize the temperature dependency of thesolubility of succinic acid; a concentration-crystallization method inwhich succinic acid is precipitated out by evaporating a solvent from asolution by heating, pressure reduction or the like to thereby increasethe concentration of succinic acid in the solution; a poor solventcrystallization method in which succinic acid is precipitated out byadding a third component (poor solvent), which reduces the solubility ofsuccinic acid, to a succinic acid-containing solution; and a combinationof these methods.

Further, in cases where the succinic acid-containing solution alsocontains a salt of succinic acid, crystals of succinic acid can beformed by adding a strong acid such as sulfuric acid or hydrochloricacid to the succinic acid-containing solution so as to convert the saltof succinic acid into succinic acid of non-dissociated form and thenperforming the above-described methods, such as cooling, concentrationand addition of a poor solvent, in combination.

As for the cooling crystallization, examples of its cooling methodinclude a method in which a succinic acid-containing solution is cooledby allowing it to circulate through an external heat exchanger or thelike; a method in which a tube (inner coil) through which a coolantpasses through is put into a succinic acid-containing solution; and amethod in which the internal pressure of an apparatus is reduced toallow a solvent contained in a solution to be vaporized and cool thesolution by means of the vaporization heat of the solvent. Thereamong,the method in which the internal pressure of an apparatus is reduced toallow a solvent contained in a solution to be vaporized and cool thesolution by means of the vaporization heat of the solvent is preferredbecause not only inhibition of heat transfer, which is caused bysuccinic acid precipitating at the heat exchange interface, can beprevented, but also succinic acid can be concentrated in the solution.This method is preferred also from the standpoint of crystallizationyield.

The concentration of succinic acid contained in the succinicacid-containing solution to be supplied to a crystallization bath ispreferably 10% by weight to 45% by weight, more preferably 15% by weightto 40% by weight, particularly preferably 20% by weight to 35% byweight.

As a crystallization solvent, for example, water; an organic acid suchas acetic acid or propionic acid; an ester such as ethyl acetate; analcohol such as methanol, ethanol, propanol, isopropanol, butanol,2-ethyl-1-hexanol or isobutanol; an ether such as diethyl ether,di-n-butyl ether, diisopropyl ether, di-n-butyl ether, tetrahydrofuranor dioxane; a ketone such as acetone, methyl ethyl ketone or diethylketone; a nitrile such as acetonitrile; or a mixture of these solventscan be employed. Thereamong, water is most preferred. As water, forexample, deionized water, distilled water, river water, well water ortap water is usually used.

The temperature of a succinic acid-containing fluid in thecrystallization bath at the time of crystallization (hereinafter, may bereferred to as “the crystallization temperature”) is set to be atemperature at which succinic acid is crystallized from the succinicacid-containing fluid and it is usually 5° C. to 60° C., preferably 10°C. to 50° C. When the crystallization temperature is lower than 5° C.,although succinic acid can be obtained in a high yield, an extremelylarge equipment is required for cooling. In addition, in a method wherecooling is performed by circulating a coolant through a jacket or aninner coil, the difference in the temperature between succinic acidslurry and the coolant becomes excessively large at the heat-transfersurface to cause severe scaling, which is not desired. Thus, in themethod where cooling is performed by circulating a coolant through ajacket or an inner coil, from the standpoint of preventing scaling, itis desired that the difference in the temperature between the succinicacid slurry and the coolant at the heat-transfer surface be set to beusually not larger than 20° C., preferably not larger than 10° C.

Further, the internal pressure of the crystallization bath to be used inthe method in which crystallization is performed under reduced pressureis determined in accordance with the desired crystallizationtemperature, and it is usually 0.5 kPa to 20 kPa, preferably 1 kPa to 15kPa, particularly preferably 1.5 kPa to 10 kPa. When the pressure islow, the temperature inside the crystallization bath can be furtherlowered and the crystallization efficiency can thus be improved;however, depending on the concentration of the succinic acid-containingfluid to be supplied, the slurry concentration in the crystallizationbath may become excessively high, so that the handling thereof and/orpressure control may become difficult. Furthermore, the equipment to beused for pressure reduction is limited and the equipment cost is thusincreased in general, which is not economically preferred. For example,in cases where steam ejectors are used for pressure reduction, anincrease in the degree of pressure reduction leads to an increase in theequipment cost due to, for example, the necessity for increasing thenumber of the steam ejectors. On the other hand, when the pressure ishigh, the temperature inside the crystallization bath becomes high, sothat the slurry concentration in the crystallization bath becomesexcessively low to impair the crystallization efficiency. Avacuum-generating device can be selected from known means in accordancewith the desired pressure as well as the presence or absence of asolvent to be evaporated with water, the type of the solvent and thelike. Examples of the known means include those which are described inDesign and Operation Series No. 3, revised, Crystallization (Sekkei-SosaSeries No. 3, kaitei, Shoseki; Kagaku Kogaku Sha, p. 292-293), such aswater, steam ejectors and oil-sealed rotary vacuum pumps.

The crystallization operation may be performed in a batch system where,after loading the total amount of the succinic acid-containing fluid tobe crystallized into the crystallization bath, the fluid is subjected tocrystallization and then the total amount of the resulting fluid isrecovered. Alternatively, the crystallization operation may be performedin a continuous system where, in order to prevent the crystallizationbath from being emptied during the crystallization operation, thesuccinic acid-containing fluid is supplied and removed as appropriatewhile performing the crystallization operation.

In cases where crystallization is performed in a continuous system, inorder to prevent the crystallization bath from being emptied, the supplyof the succinic acid-containing fluid to the crystallization bath iscarried out by, for example, a method in which the fluid is continuouslyor intermittently supplied with pressure by utilizing a liquid feed pumpor a pressure difference. The succinic acid solution is usually suppliedat such a rate at which the average residence time thereof in thecrystallization bath is 0.5 hour to 10 hours. When the residence time inthe crystallization bath is short, the supersaturation degree ofsuccinic acid in the bath is increased, so that microcrystals are formedin a large amount. In addition, since the slurry is removed from thecrystallization bath while maintaining a degree of supersaturation,there may arise problems of scaling and the like in the subsequentsteps. Meanwhile, an excessively long residence time is also inefficientsince it requires an unnecessarily large crystallization bath.

In cases where crystallization is performed in a continuous system,usually, the resulting succinic acid is recovered from thecrystallization bath in the form of a slurry along with the succinicacid-containing fluid. In this case, the recovery is carried out by, forexample, a method in which a slurry is, by means of pressurized transferor the like which utilizes a pressure difference, continuously orintermittently recovered into a receiver tank having a pressure lowerthan that of the slurry pump or crystallization bath, whileappropriately comparing the recovered amount and the supplied amount ofthe succinic acid-containing fluid so that the crystallization bath doesnot become empty.

In order to supply and recover the succinic acid-containing fluid to andfrom the crystallization bath in such a manner that the crystallizationbath does not become empty, for example, a method in which the amount ofsupply and that of recovery are controlled to be the same or a method inwhich a fluid level sensor or the like is used to repeat an operation ofsupplying the fluid when the fluid level in the crystallization bath hasdeclined to a certain level and an operation of removing the fluid whenthe fluid level has risen to a certain level can be employed.

In the crystallization operation, in order to control the particle sizedistribution of the resulting succinic acid crystals, it is desired thatnucleation and crystal growth of succinic acid be controlled. Thenucleation and crystal growth of succinic acid is usually controlled byadjusting the supersaturation degree of succinic acid in the bath and,for this purpose, a method in which the crystallization time iscontrolled is usually employed. The crystallization time is usually 0.5hour to 10 hours, preferably 1 hour to 5 hours. For example, in caseswhere cooling crystallization operation is performed in a batch system,it is desired that the succinic acid-containing fluid be cooled to aprescribed temperature over a period of 0.5 hour to 5 hours and thenaged for a period of 0.1 hour to 5 hours. In this process, the rate atwhich the succinic acid-containing fluid is cooled is usually 0.05°C./min to 2° C./min, preferably 0.1° C./min to 1.5° C./min, particularlypreferably 0.2° C./min to 1° C./min. Further, in the cases where coolingcrystallization operation is performed in a continuous system, it isdesired that the average residence time of the succinic acid-containingfluid be 0.5 hour to 10 hours, preferably 1 hour to 5 hours. When thecrystallization time or the average residence time is short, thesupersaturation degree of succinic acid in the bath is increased and thenucleation rate becomes high, so that microcrystals are formed in alarge amount. In addition, since the slurry is removed from thecrystallization bath while maintaining a degree of supersaturation,there may arise problems of scaling and the like in the subsequentsteps.

Meanwhile, an excessively long crystallization time is also inefficientsince it requires an unnecessarily large crystallization bath.

(Crystallization Bath)

The constitution of the crystallization bath is not particularlyrestricted as long as it is a vessel equipped with a stirring device. Asthe crystallization bath, a vessel having a conventionally knownstirring device may be employed; however, a cylindrical vessel having abottom is preferably employed. Further, in order to attain efficientshearing of the slurry, it is preferred that baffle plates be arrangedinside the bath. Moreover, in order to control the flow inside thecrystallization bath, a vessel having a cylindrical guide such as aguide called “draft tube” can also be employed.

The shape of the vessel is not particularly restricted; however, inorder to make the slurry more uniform in the apparatus and to attainmore efficient shearing of the slurry, the vessel has a ratio betweenthe diameter and the height (L/D) of usually 0.5 to 3, preferably 0.7 to2.5, particularly preferably 1 to 2.

(Stirring Device)

As a stirring device, one which is equipped with a stirring blades isemployed. The stirring blade is not required to be a special blade andany known stirring blade can be used. The stirring blade is selectedfrom, for example, shearing-type blades such as paddle blades andturbine blades; and discharge-type blades such as sweep-back blades,Pfaudler impellers, Maxblend Blade (registered trademark; Sumitomo HeavyIndustries, Ltd.) and Fullzone Blade (registered trademark; ShinkoPantec Co., Ltd.).

The size of the stirring blade is also not particularly restricted, andanother stirring device which circulates the fluid may also be used incombination of the stirring device equipped with a stirring blade.

As such stirring device, for example, a device which allows the succinicacid-containing solution transferred from the crystallization bath to becirculated back to the crystallization bath by means of a fluid transferpump such as a centrifugal pump can be employed.

The stirring of the crystallization bath is not particularly restrictedas long as the crystals of succinic acid are kept flowing withoutsettling in the crystallization bath. From the standpoints that thepurity of the resulting succinic acid can be improved and that succinicacid crystals having a more uniform particle size can be obtained,crystallization is performed under such stirring conditions where thepower required for stirring a unit volume of the succinicacid-containing solution (hereinafter, may be abbreviated as “Pv”) is0.2 kW/m³ to 5 kW/m³, preferably 0.4 kW/m³ to 3 kW/m³.

(Solid-Liquid Separation)

After the crystallization, the resulting succinic acid slurry can besubjected to a solid-liquid separation treatment in accordance with aknown method to separate succinic acid crystals from the mother liquor.The separation method is not particularly restricted and examplesthereof include filtration separation and sedimentation separation.Further, the separation operation may be performed either batchwise orcontinuously. Examples of an efficient solid-liquid separator includecontinuous-type centrifugal filtration apparatuses and centrifugalsettlers such as decanters. In addition, depending on the desired purityof succinic acid, a wet cake recovered by the solid-liquid separationmay be rinsed with cold water or the like.

(Drying)

The succinic acid recovered by crystallization can be dried by aconventional method depending on the application thereof. Generally, thesuccinic acid is dried to a water content of usually not less than 0.1%by weight, preferably not less than 0.2% by weight, and on anotherfront, usually not higher than 2% by weight, preferably not higher than1%. The drying method is not particularly restricted and, for example, aconvection heating-type dryer such as a band dryer or a fluidized-beddryer, or a conductive heat transfer-type dryer such as a drum dryer canbe employed. A fluidized-bed dryer is particularly preferred since it iscapable of performing a continuous treatment in a large quantity and thebreakage of crystals in the process of drying treatment is limited.Further, when a fluidized-bed dryer is used, from the standpoint ofpreventing the succinic acid from causing dust explosion, it ispreferred that the drying be performed under nitrogen supply and thatthe atmosphere be controlled to have an oxygen concentration of nothigher than 12%. Moreover, since succinic anhydride may be generated asa result of intramolecular dehydration, the succinic acid is dried sothat the temperature thereof during the drying process is maintained atpreferably not higher than 100° C., more preferably not higher than 90°C., still more preferably not higher than 80° C.

Specific examples of a crystallization apparatus include the one shownin FIG. 4 which comprises an aqueous succinic acid solution feed tank; acrystallization bath having two rows of four baffle plates and fourinclined paddle blades (stirring blades); a slurry recovery tank and thelike.

<Activated Carbon Treatment>

In cases where an activated carbon treatment is performed, as theactivated carbon to be used, any known activated carbon such as acoal-based activated carbon, a wood-based activated carbon, a coconutshell-based activated carbon or a resin-based activated carbon can beemployed. In addition, activated carbons that are obtained by activatinga variety of these material activated carbons, such as coal-based,wood-based, coconut shell-based and resin-based activated carbons, inaccordance with a method such as a gas activation method, a steamactivation method or a chemical activation method using zinc chloride,phosphoric acid or the like, can also be used.

Specific examples of such activated carbon include Calgon CPG, CalgonCAL, Calgon SGL, Diasorb W, Diahope MS10, Diahope M010, Diahope MS16,Diahope 6MD, Diahope 6MW, Diahope 8ED, Diahope ZGN4 and Centur, all ofwhich are manufactured by Calgon-Mitsubishi Chemical Corporation; GAC,GAC PLUS, GCN PLUS, C GRAN, RO, ROX, DARCO, CN, SX, SXPLUS, SA, SX, PKand W, all of which are manufactured by Norit Japan Co., Ltd.; GW, GWH,GLC, 4GC, KW, PW and PK, all of which are manufactured by KurarayChemical Co., Ltd.; HC-30S, GL-30S, 4G-3S, PA and PC, all of which aremanufactured by Tsurumicoal Co., Ltd.; P, W, CW, SG, SGP, S, GB, CA andK, all of which are manufactured by Futamura Chemical Co., Ltd.;Shirasagi KL, Shirasagi W2C, Shirasagi WH2C, Shirasagi W5C, ShirasagiWH5C, Shirasagi WH5X, Shirasagi XS7100H-3, Carboraffin, Shirasagi A,Shirasagi C and Shirasagi M, all of which are manufactured by JapanEnviroChemicals Ltd.; and Hokuetsu CL-K, Hokuetsu HS and Hokuetsu KS,all of which are manufactured by Ajinomoto Fine-Techno Co., Inc.

Among these activated carbons, coconut shell-based activated carbons andwood-based activated carbons are preferred since they are capable ofefficiently removing impurities showing absorption in the ultravioletregion of 250 to 300 nm that are contained in succinic acid. Meanwhile,from the standpoint of efficiently removing a color component ofsuccinic acid, activated carbons that are obtained by a method such as agas activation method, a steam activation method or a chemicalactivation method using zinc chloride, phosphoric acid or the like arepreferred. Thereamong, activated carbons that are obtained by a steamactivation method or a chemical activation method using zinc chloride,phosphoric acid or the like are more preferred and activated carbonsthat are activated with a chemical agent such as zinc chloride orphosphoric acid are particularly preferred. The shape of the activatedcarbon to be used may take any of a powder form, a crushed form, amolded form and a fibrous form. In cases where the activated carbon isloaded into a column, from the standpoint of controlling the columnpressure, the activated carbon is preferably in the form of particles orgranules.

As a method of the activated carbon treatment, either a method in whicha succinic acid-containing solution is mixed with an activated carbon ina batchwise manner and the resulting mixture is then separated byfiltration or a method in which the solution is passed through a packedbed of an activated carbon can be employed. In cases where a batchsystem is used, the treatment time is usually not shorter than 5minutes, preferably not shorter than 10 minutes, and on another front,usually not longer than 5 hours, preferably not longer than 2 hours. Incases where a packed bed system is used, the treatment time is, in termsof SV (space velocity), usually 0.1 hr⁻¹ to 20 hr⁻¹. The treatmenttemperature is usually 20° C. to 90° C. As described in the above, thetype of impurities to be removed is different depending on the type ofthe activated carbon; therefore, as a method of removing the impurities,for example, a method in which a plural types of activated carbons areused in combination or a method in which an activated carbon treatmentis performed in combination with the above-described crystallizationtreatment, the below-described hydrogenation treatment and/or thebelow-described ion-exchange column treatment can be employed.

Further, in cases where water is used as a solvent, a succinic acidsolution derived from fermentation may contain a component insoluble towater. Inclusion of such an insoluble component causes a reduction inthe efficiency of removing the above-described impurities by anactivated carbon and the subsequent purification step; therefore, it ispreferred that such an insoluble component be removed in advance.Removal of the insoluble component is preferably carried out by a methodin which, in-between the step of deriving succinic acid from a generatedsalt of succinic acid by a fermentation method and the step ofperforming an activated carbon treatment, the insoluble component isremoved from a succinic acid solution originated from fermentation bysubjecting the solution to a known membrane permeation treatment.Alternatively, a method in which the permeability of a membranepermeation treatment is improved by allowing the insoluble component tobe adsorbed under the coexistence of powder-form activated carbon or amethod in which an appropriate powder-form activated carbon is used toadsorb and remove the insoluble component simultaneously with theabove-described impurities can also be suitably employed.

Further, in the present invention, in cases where removal of impuritiesis carried out by performing a crystallization treatment and/or anactivated carbon treatment in combination with a hydrogenationtreatment, for example, but not particularly limited to, a process inwhich the step of crystallization and/or activated carbon treatment iscarried out prior to the below-described hydrogenation treatment step issuitably employed since such a process efficiently removes theimpurities.

<Hydrogenation Treatment>

Succinic acid obtained by a microbial reaction usually contains an odorcomponent. It is preferred that the amount of the odor component in thesuccinic acid be reduced.

As a method for removing an odor component, there are known, forexample, a deodorization method which utilizes an adsorbent such as anactivated carbon; a method in which an odor component is removed bywashing with an organic solvent; a crystallization method; and anaeration method. For removal of an odor component, a hydrogenationtreatment in the presence of a catalyst is particularly effective.Meanwhile, a succinic acid-containing solution derived from a biomassresource by fermentation or the like may contain a small amount offumaric acid.

When a succinic acid-containing solution derived from a biomass resourceby fermentation or the like is subjected to a hydrogenation treatment,not only the odor component contained in succinic acid can be easilyremoved, but also, in cases where the solution contains fumaric acid asdescribed above, succinic acid is generated from fumaric acid and theyield of succinic acid can thus be improved at the same time; therefore,as a method for deodorizing succinic acid, such hydrogenation treatmentmethod is an exceptional technique as compared to conventional methods.It is preferred that the purification process include the step ofsubjecting a succinic acid-containing solution derived from fermentationto a hydrogenation treatment in the presence of a catalyst.

The hydrogenation treatment may take either a batchwise reaction systemor a continuous reaction system and can be performed in accordance witha conventionally known method. Specific examples of the hydrogenationtreatment method include a method in which, after allowing a succinicacid-containing solution and a hydrogenation catalyst to coexist in apressurized reactor and subjecting this mixture to a hydrogenationtreatment with stirring by introducing a hydrogen gas thereto, the thustreated succinic acid-containing reaction solution is separated from thehydrogenation catalyst and recovered from the reactor; a method in whicha hydrogenation treatment is performed using a fixed-bed multi-tubularreactor or a single-tubular reactor while circulating a succinicacid-containing solution and hydrogen gas from a lower section of thereactor and the thus treated succinic acid-containing reaction solutionis then recovered; and a method in which a hydrogenation treatment isperformed by introducing a hydrogen gas from a lower section of areactor and circulating a succinic acid-containing solution from anupper section of the reactor and the thus treated succinicacid-containing reaction solution is then recovered.

As the hydrogenation catalyst, a known homogeneous or heterogeneousnoble metal-containing hydrogenation catalyst can be employed. Specificexamples of such hydrogenation catalyst include, but not particularlylimited to, those hydrogenation catalysts containing a noble metal suchas ruthenium, rhodium, palladium or platinum. Thereamong, hydrogenationcatalysts containing palladium or platinum, particularly palladium, arepreferred.

These hydrogenation catalysts may be used as-is in the form of acompound containing the above-described noble metal or may be used in aform in which a ligand such as organic phosphine is allowed to coexist;however, from the standpoint of the easiness of catalyst separation, aheterogeneous noble metal-containing catalyst is preferred.

Further, a hydrogenation treatment can be performed by using such anoble metal-containing compound in coexistence with a metal oxide suchas silica, titania, zirconia or activated alumina, a complex metal oxidethereof or an activated carbon. This method is preferred because, notonly an odor component contained in succinic acid derived fromfermentation, but also color components and organic impurities can besimultaneously adsorbed and removed, so that efficient removal ofimpurities can be achieved. The same effects can be attained also byusing a catalyst prepared by supporting the above-described noble metalon a carrier such as a metal oxide (e.g., silica, titania, zirconia oractivated alumina), a complex metal oxide thereof or an activatedcarbon; therefore, a method in which such a supported catalyst is usedcan also be suitably employed. The amount of the noble metal to besupported is usually 0.1 to 10% by weight of the carrier. The carrier isnot particularly restricted; however, it is preferably silica or anactivated carbon, particularly preferably an activated carbon, since theamount of the metal eluting therefrom during the hydrogenation treatmentis small.

Accordingly, in the present invention, the embodiment in which ahydrogenation treatment is performed with a hydrogenation catalystprepared by supporting a noble metal on a carrier such as a metal oxide(e.g., silica, titania, zirconia or activated alumina), a complex metaloxide thereof or an activated carbon is included in the definition of anembodiment in which a hydrogenation treatment is performed with ahydrogenation catalyst in the presence of an adsorbent selected from thegroup consisting of metal oxides, silica and activated carbons.

As a solvent into which succinic acid derived from a biomass resource isincorporated at the time of the hydrogenation treatment, water; anorganic acid such as acetic acid or propionic acid; an ester such asethyl acetate; an alcohol such as methanol, ethanol, propanol,isopropanol, butanol, 2-ethyl-1-hexanol or isobutanol; an ether such asdiethyl ether, di-n-butyl ether, diisopropyl ether, di-n-butyl ether,tetrahydrofuran or dioxane; a ketone such as acetone, methyl ethylketone or diethyl ketone; a nitrile such as acetonitrile; or a mixtureof these solvents can be employed. Thereamong, water is most preferred.As water, for example, deionized water, distilled water, river water,well water or tap water is usually used. As required, a solution, whichis obtained as a result of the step of crystallizing succinic acid froma succinic acid-containing reaction solution after hydrogenationreaction and the subsequent filtration, can also be repeatedly used. Thesolution may have any succinic acid concentration as long as it is nothigher than the saturation solubility at the solution temperature.

The hydrogen gas to be used may be pure hydrogen; however, hydrogendiluted with an inert gas such as nitrogen, helium or argon can also beused. In consideration of the effect on the efficiency of thehydrogenation treatment, the concentration of carbon monoxide in thehydrogen gas is usually not higher than 10,000 ppm, preferably nothigher than 2,000 ppm, more preferably not higher than 1,000 ppm.

As for the hydrogen pressure in the hydrogenation treatment, when it isexcessively low, the reaction rate becomes slow, so that a long time isrequired to complete the reaction. Meanwhile, when the hydrogen pressureis excessively high, depending on the catalyst and reaction conditions,hydrides of succinic acid such as butanediol and tetrahydrofuran aregenerated as by-products. Therefore, the hydrogen pressure in thehydrogenation treatment is usually not lower than 0.1 MPa, and the upperlimit thereof is usually not higher than 5 MPa, preferably not higherthan 3 MPa, more preferably not higher than 1 MPa.

As for the temperature of the hydrogenation treatment, when it isexcessively low, the reaction rate becomes slow, so that a long time isrequired to complete the reaction. Meanwhile, when the temperature isexcessively high, hydrides of succinic acid are generated as by-productsand the amount of by-products such as malic acid is increased when wateris used as the solvent. Therefore, the temperature of the hydrogenationtreatment is usually not lower than 30° C., preferably not lower than50° C., and the upper limit thereof is usually not higher than 150° C.,preferably not higher than 120° C.

<Ion-Exchange Column Treatment>

Further, in the present invention, in order to remove impuritiescontained in succinic acid, a purification operation such as anion-exchange column treatment may also be performed in combination.

The term “ion-exchange column treatment” used herein refers to a processof removing an ion by treating the liquid to pass through a columnloaded with an ion-exchange resin. The ion-exchange resin should beselected in accordance with the ions contained in the liquid to betreated and the required purity of succinic acid. For example, in orderto remove an anion such as sulfate ion or chloride ion, ananion-exchange resin (OH-type) can be employed and, in order to remove acation such as a metal ion or ammonium ion, a cation-exchange resin(H-type) can be employed. These ion-exchange resins may also be used incombination as required.

Ion-exchange resins are classified into strongly acidic cation-exchangeresins, weakly acidic cation-exchange resins, strongly basicanion-exchange resins and weakly basic anion-exchange resins, based onthe strength of the functional group as an acid or a base. Further,based on the form thereof, ion-exchange resins are also classified intogel-type and porous-type. In the present invention, the ion-exchangeresin to be used is not particularly restricted. However, taking intoconsideration the ion exchange efficiency, it is preferred to use astrongly acidic cation-exchange resin having a higher strength as anacid and/or a strongly basic anion-exchange resin having a higherstrength as a base. Moreover, since there is no particular reason thatthe ion-exchange resin has to be a porous-type, it is desired to use amore versatile and inexpensive gel-type ion-exchange resin. Specificexamples of such cation-exchange resin include Diaion SK1B (H-type) andspecific examples of such anion-exchange resin include Diaion SA10A.

The ion-exchange column treatment can be performed within thetemperature range which is not lower than the temperature at whichsuccinic acid is dissolved in the liquid to be treated but lower thanthe heat-resistant temperature of the ion-exchange resin. That is, incases where a cation-exchange resin is used, the ion-exchange columntreatment is usually performed at a temperature of 20 to 100° C.,although this is variable depending on the concentration of succinicacid in the liquid to be treated. Meanwhile, since an anion-exchangeresin has a lower heat resistance as compared to a cation-exchangeresin, in cases where an anion-exchange resin is used, the ion-exchangecolumn treatment is usually performed at a temperature of 10 to 80° C.From the standpoint of the treatment temperature, in cases where ananion-exchange column treatment is performed, it is desired that a stepin which a column treatment can be performed at a low succinic acidconcentration and a low temperature be adopted.

Further, the method of allowing a liquid to pass through a column fortreatment is not particularly restricted. When the throughput rate isexcessively high, the pressure loss before and after the column isincreased and the ion exchange is not sufficiently performed, while whenthe throughput rate is needlessly slow, an unnecessarily large column isrequired. Therefore, the treatment is usually performed at a spacevelocity (SV) of 0.1 to 10 hr⁻¹ and a superficial velocity of 1 to 20m/hr.

Usually, in a column treatment, the ion concentration is measured at thecolumn outlet at all times or at regular intervals and, if ion leakagewere detected at the column outlet, the ion-exchange resin is subjectedto a regeneration treatment. Regeneration of the ion-exchange resin canbe carried out in accordance with a conventional method by using an acidsuch as sulfuric acid or hydrochloric acid in the case of acation-exchange resin or an alkali such as caustic soda in the case ofan anion-exchange resin.

The organic acid such as succinic acid obtained by the production methodof the present invention contains a small amount of the above-describedimpurities such as an aromatic carboxylic acid, and the organic acid hasan average absorbance of preferably not higher than 0.05, morepreferably not higher than 0.03, particularly preferably not higher than0.01, in the ultraviolet region of 250 to 300 nm. When succinic acidhaving a high average absorbance is used as a starting material of apolyester, a markedly colored polymer is produced.

In the present invention, the term “absorbance” refers to a value whichis obtained by measuring a 3.0%-by-weight aqueous succinic acid solutionplaced in a quartz cell of 1 cm in optical path length by using anultraviolet-visible absorption spectrophotometer. The measurement ofabsorbance can be performed by using a commercially availableultraviolet-visible absorption spectrophotometer.

The term “absorbance (A)” used herein means an absorbance measured at anoptical path length of 1 cm and it is calculated in accordance with thefollowing definition.

A=log 10(I ₀ /I)

(wherein, I₀ represents the intensity of incident light; and Irepresents the intensity of transmitted light.)

Further, the term “average absorbance in the ultraviolet region of 250to 300 nm” means a value which is obtained by dividing the sum ofabsorbances measured at 1 nm intervals in the wavelength range of 250 to300 nm by 51.

Average absorbance=(Sum of absorbances measured at 1 nm intervals in thewavelength region of 250 to 300 nm)/51

The concentration of protocatechuic acid is, with respect to thesuccinic acid obtained by the production method of the presentinvention, preferably not higher than 80 ppm, more preferably not higherthan 30 ppm, still more preferably not higher than 15 ppm, yet stillmore preferably not higher than 10 ppm, further still more preferablynot higher than 5 ppm, particularly preferably not higher than 3 ppm.When the concentration of protocatechuic acid is high, the coloration ofthe resulting polymer is increased. Further, in order to control theconcentration of protocatechuic acid at a low level, an excessivepurification treatment is required; however, its polymercoloration-improving effect is limited and not efficient.

The concentration of uvitonic acid is, with respect to the succinic acidobtained by the production method of the present invention, preferablynot higher than 300 ppm, more preferably not higher than 150 ppm, stillmore preferably not higher than 100 ppm, yet still more preferably nothigher than 30 ppm, particularly preferably not higher than 10 ppm. Whenthe concentration of uvitonic acid is high, the coloration of theresulting polymer is increased. Further, in order to control theconcentration of protocatechuic acid at a low level, an excessivepurification treatment is required; however, its polymercoloration-improving effect is limited and not efficient.

Here, also in cases where an organic acid other than succinic acid isproduced, the concentration of protocatechuic acid is, with respect tothe organic acid of interest obtained by the production method of thepresent invention, preferably not higher than 80 ppm, more preferablynot higher than 30 ppm, still more preferably not higher than 15 ppm,yet still more preferably not higher than 10 ppm, further still morepreferably not higher than 5 ppm, particularly preferably not higherthan 3 ppm. Moreover, the concentration of uvitonic acid is, withrespect to the organic acid of interest obtained by the productionmethod of the present invention, preferably not higher than 300 ppm,more preferably not higher than 150 ppm, still more preferably nothigher than 100 ppm, yet still more preferably not higher than 30 ppm,particularly preferably not higher than 10 ppm.

Further, it is usually preferred that the succinic acid produced in thepresent invention have a low absorbance in the visible light region anda low level of coloration. As for the yellowness (Y.I. value) of thesuccinic acid, the upper limit thereof is usually not higher than 50,preferably not higher than 30, more preferably not higher than 20, stillmore preferably not higher than 10, yet still more preferably not higherthan 6, particularly preferably not higher than 4, while the lower limitthereof is, though not particularly restricted, usually not lower than−10, preferably not lower than −5, more preferably not lower than −1.When succinic acid showing a high Y.I. value is used as a startingmaterial of a polymer, there is a drawback that a markedly coloredpolymer is produced. Meanwhile, succinic acid showing a low Y.I. valueis a more preferred mode; however, from the economical standpoint, suchsuccinic acid has disadvantages in that, for example, the productionthereof requires a huge investment to be made in equipments and isconsiderably time-consuming. In the present invention, the Y.I. value ismeasured by the method according to JIS K7105.

The organic acid such as succinic acid obtained by the production methodof the present invention may contain, as an impurity, elemental nitrogenwhich is originated from a biomass resource or generated by afermentation treatment or a purification treatment including a step ofperforming neutralization with an acid. More specifically, the organicacid obtained by the production method of the present invention maycontain elemental nitrogen originated from an amino acid, a protein, anammonium salt, urea, a fermentative bacterium or the like.

The upper limit of the nitrogen atom content in the organic acid such assuccinic acid obtained by the production method of the present inventionis, in terms of the amount of atoms, usually not higher than 2,000 ppm,preferably not higher than 1,000 ppm, more preferably not higher than100 ppm, most preferably not higher than 20 ppm. The lower limit of thenitrogen atom content is usually not less than 0.01 ppm, preferably notless than 0.05 ppm, and from the standpoint of economical efficiency ofthe purification step, more preferably not less than 0.1 ppm, still morepreferably not less than 1 ppm.

The nitrogen atom content is measured by a known method such as anelemental analysis method or a method in which, after separating anamino acid or ammonia from a sample under a biological amino acidseparation condition using an amino acid analyzer, the thus separatedamino acid or ammonia is subjected to ninhydrin coloration and thendetected.

In cases where succinic acid having a nitrogen atom content in theabove-described range is used as a starting material of a polyester, thecoloration of the resulting polyester is reduced, which is advantageous.In addition, the use of such succinic acid also has an effect ofinhibiting a delay in the polymerization reaction of polyester.

Further, the organic acid such as succinic acid obtained by theproduction method of the present invention may also contain a sulfuratom generated by, for example, a purification treatment which includesa step of performing neutralization with an acid. Specific examples ofimpurities containing a sulfur atom include sulfuric acid, sulfates,sulfurous acid, organic sulfonic acids and organic sulfonates.

The sulfur atom content in the organic acid such as succinic acidobtained by the production method of the present invention is notparticularly restricted; however, when it is excessively high, the useof the organic acid as a starting material of a polyester tends tocause, for example a delay in the polymerization reaction, partialgelation of the generated polymer and a reduction in the stability ofthe generated polymer. On the other hand, an excessively low sulfur atomcontent makes the purification step complicated. Therefore, the upperlimit of the sulfur atom content in a dicarboxylic acid is, in terms ofthe amount of atoms, usually not higher than 100 ppm, preferably nothigher than 20 ppm, more preferably not higher than 10 ppm, particularlypreferably not higher than 5 ppm, most preferably not higher than 0.5ppm. Meanwhile, the lower limit of the sulfur atom content is usuallynot less than 0.001 ppm, preferably not less than 0.01 ppm, morepreferably not less than 0.05 ppm, particularly preferably not less than0.1 ppm. Here, the sulfur atom content is measured by a known elementalanalysis method.

The organic acid such as succinic acid obtained by the production methodof the present invention may contain an alkali metal element. When thealkali metal content in an aliphatic dicarboxylic acid is excessivelyhigh, the use thereof as a starting material of a polymer not onlyreduces the thermal stability and hydrolysis resistance, but also causesa severe inhibition of polymerization during polymerization, so that apolymer of a high polymerization degree which has practically sufficientmechanical characteristics may not be obtained. Therefore, the alkalimetal content is usually not higher than 50 ppm, preferably not higherthan 30 ppm, more preferably not higher than 10 ppm, particularlypreferably not higher than 5 ppm.

<Method of Producing Polymer>

Further, in the present invention, after producing an organic acid suchas succinic acid in accordance with the above-described method, byperforming a polymerization reaction using the thus obtained organicacid as a starting material, an organic acid-containing polymer can beproduced. In recent years, with an increasing number ofenvironment-friendly industrial products, polymers prepared from amaterial of plant origin have been drawing attention. Particularly, thesuccinic acid produced in the present invention can be processed intoknown polymers that are produced by using a dicarboxylic acid component,preferably an aliphatic dicarboxylic acid as a starting material, suchas polyesters, polyamides and polyurethanes. Specific examples ofsuccinic acid unit-containing polymers include polyesters that areobtained by polymerization between a diol such as butanediol or ethyleneglycol and succinic acid; and polyamides that are obtained bypolymerization between a diamine such as hexamethylenediamine andsuccinic acid.

As one example of the method of producing a polymer, a method ofproducing a polyester will now be described.

<Method of Producing Polyester>

In the present invention, as a method of producing a polyester, aconventionally known method can be employed. For example, a polyestercan be produced by a commonly used melt-polymerization method in whichan esterification reaction and/or a transesterification reaction iscarried out between an aliphatic dicarboxylic acid component containingthe above-described succinic acid and a diol component and the resultantis then subjected to a polycondensation reaction under reduced pressureor by a known solution-heating dehydration condensation method in whichan organic solvent is used; however, from the standpoint of economicalefficiency and simplicity of the production process, a method in which apolyester is produced by melt polymerization in the absence of solvent.

<Dicarboxylic Acid Component>

The dicarboxylic acid component is not particularly restricted as longas it contains the succinic acid obtained by the above-described methodof producing an organic acid. The dicarboxylic acid component maycontain an aliphatic and/or aromatic dicarboxylic acid derived from afossil resource; however, it is preferred that the dicarboxylic acidcomponent contain the succinic acid obtained by the above-describedmethod of producing an organic acid.

<Diol Component>

The diol component is not particularly restricted; however, it ispreferably an aliphatic diol.

The aliphatic diol is not particularly restricted as long as it is analiphatic or alicyclic compound having two OH groups, and examplesthereof include aliphatic diols in which the lower limit of the numberof carbon atoms is not less than 2 and the upper limit is usually notmore than 10, preferably not more than 6.

Specific examples of such aliphatic diol include ethylene glycol,1,3-propylene glycol, neopentylglycol, 1,6-hexamethylene glycol,decamethylene glycol, 1,4-butanediol and 1,4-cyclohexanedimethanol.These aliphatic diols may be used individually, or two or more thereofmay be used in combination as a mixture.

Thereamong, ethylene glycol, 1,4-butanediol, 1,3-propylene glycol and1,4-cyclohexanedimethanol are preferred and ethylene glycol,1,4-butanediol and a mixture thereof are more preferred. Further, analiphatic diol containing 1,4-butanediol as a main component or1,4-butanediol is particularly preferred. The term “main component”means that the amount of the component is, with respect to the totalamount of diol units, usually not less than 50 mol %, preferably notless than 60 mol %, more preferably not less than 70 mol %, particularlypreferably not less than 90 mol %.

Further, a polyether terminated with a hydroxy group at both ends mayalso be used in combination with the above-described aliphatic diol. Inthe polyether terminated with a hydroxy group at both ends, the lowerlimit of the number of carbon atoms is usually not less than 4,preferably not less than 10, and the upper limit is usually not morethan 1,000, preferably not more than 200, more preferably not more than100.

Specific examples of such polyether terminated with a hydroxy group atboth ends include diethylene glycol, triethylene glycol, polyethyleneglycol, polypropylene glycol, polytetramethylene glycol,poly-1,3-propanediol and poly-1,6-hexamethylene glycol. Further, forexample, a copolymer polyester composed of polyethylene glycol andpolypropylene glycol can also be used. The amount of the polyetherterminated with a hydroxy group at both ends to be used is, in terms ofthe content in the resulting polyester, a calculated amount of usuallynot more than 90% by weight, preferably not more than 50% by weight,more preferably not more than 30% by weight.

<Other Copolymer Component>

In the production of a polyester according to the present invention, inaddition to the above-described diol component and dicarboxylic acidcomponent, a copolymer component may be added as well.

Specific examples of the copolymer component include at least onepolyfunctional compound selected from the group consisting ofbifunctional oxycarboxylic acids, unsaturated dicarboxylic acids, tri-or higher functional polyhydric alcohols for forming a cross-linkedstructure, tri- or higher functional polycarboxylic acids or anhydridesthereof and tri- or higher functional oxycarboxylic acids. When thesecopolymer components are added, an effect of considerably improving thepolymerization rate in the polyester production is exerted. Among thesecopolymer components, an oxycarboxylic acid is suitably employed sinceit tends to allow a polyester having a high polymerization degree to beeasily produced.

Specific examples of the bifunctional oxycarboxylic acids include lacticacid, glycolic acid, hydroxybutyric acid, hydroxycaproic acid,2-hydroxy-3,3-dimethylbutyric acid, 2-hydroxy-3-methylbutyric acid and2-hydroxyisocaproic acid, and these may also be in the form of an esteror lactone of an oxycarboxylic acid or a derivative of an oxycarboxylicacid polymer or the like. Further, these oxycarboxylic acids may be usedindividually, or two or more thereof may be used in combination as amixture. In cases where these oxycarboxylic acids have optical isomers,they may also be any of a D-isomer, an L-isomer and a racemic body.Moreover, these oxycarboxylic acids may be in the form of a solid, aliquid or an aqueous solution. Thereamong, readily available lactic acidand glycolic acid are particularly preferred. As for the form thereof, a30 to 95% aqueous solution is preferred since such an aqueous solutionis readily available. In this case, the lower limit of the amount of theoxycarboxylic acid to be used is, with respect to the amount of thematerial monomer, usually not less than 0.02 mol %, preferably not lessthan 0.5 mol %, more preferably not less than 1.0 mol %, while the upperlimit thereof is usually not more than 30 mol %, preferably not morethan 20 mol %, more preferably not more than 10 mol %.

Examples of the unsaturated dicarboxylic acids include itaconic acid,aconitic acid, fumaric acid and maleic acid. These unsaturateddicarboxylic acids may be used individually, or two or more thereof maybe used in combination as a mixture. Since an unsaturated dicarboxylicacid causes generation of a gel, the amount of the unsaturateddicarboxylic acid to be used is, with respect to the total amount ofmonomer units constituting the polyester, usually not more than 5 mol %,preferably not more than 0.5 mol %, more preferably not more than 0.05mol %.

Specific examples of the tri- or higher functional polyhydric alcoholsinclude glycerin, trimethylolpropane and pentaerythritol. These tri- orhigher functional polyhydric alcohols may be used individually, or twoor more thereof may be used in combination as a mixture.

Specific examples of the tri- or higher functional polycarboxylic acidsor anhydrides thereof include propanetricarboxylic acids, pyromelliticacid anhydrides, benzophenonetetracarboxylic acid anhydrides andcyclopentatetracarboxylic acid anhydrides, and these may be usedindividually, or two or more thereof may be used in combination as amixture.

Specific examples of the tri- or higher functional oxycarboxylic acidsinclude malic acid, hydroxyglutaric acid, hydroxymethylglutaric acid,tartaric acid, citric acid, hydroxyisophthalic acid andhydroxyterephthalic acid. These tri- or higher functional oxycarboxylicacids may be used individually, or two or more thereof may be used incombination as a mixture. In particular, from the standpoint ofavailability, malic acid, tartaric acid and citric acid are preferred.

Since the above-described tri- or higher functional compounds causegeneration of a gel, the amount thereof to be used is, with respect tothe total amount of monomer units constituting the polyester, usuallynot more than 5 mol %, preferably not more than 0.5 mol %, morepreferably not more than 0.2 mol %.

For the conditions of temperature, time, pressure and the like,conventionally known ranges can be adopted.

With regard to the temperature of the esterification reaction and/or thetransesterification reaction performed between a succinicacid-containing aliphatic dicarboxylic acid component and a diolcomponent, the lower limit is usually not lower than 150° C., preferablynot lower than 180° C., and the upper limit is usually not higher than260° C., preferably not higher than 250° C. As for the reactionatmosphere, these reactions are usually performed under an inert gasatmosphere such as nitrogen or argon. The reaction pressure is usuallyin normal pressure to 10 kPa, preferably normal pressure.

The reaction time is usually not shorter than 1 hour and the upper limitthereof is usually not longer than 10 hours, preferably not longer than4 hours.

In the subsequent polycondensation reaction, when the pressure at thetime of producing a polyester by polymerization is excessively high, thetime required for the production is extended and, as a result, areduction in the molecular weight and coloration of the resultingpolyester are caused by thermal decomposition, so that it tends tobecome difficult to produce a polyester which exhibits practicallysufficient characteristics. Meanwhile, from the standpoint of improvingthe polymerization rate, a method in which a polyester is produced byusing an ultra-high vacuum polymerization equipment is a preferredembodiment; however, not only a huge investment needs to be made intoequipments, but also the time required for a polyester to be produced bypolymerization still tends to be long; therefore, there are concerns fora reduction in the molecular weight and coloration of the resultingpolyester that are caused by thermal decomposition. Accordingly, thepolycondensation reaction is performed at a vacuum degree of, as thelower limit, usually not lower than 0.01×10³ Pa, preferably not lowerthan 0.01×10³ Pa and, as the upper limit, usually not higher than1.4×10³ Pa, preferably not higher than 0.4×10³ Pa.

As for the reaction temperature, when it is excessively low, the rate ofpolymerization reaction becomes extremely slow, so that not only thetime required for the production of a polyester having a highpolymerization degree is extended, but also a high-power stirringmachine becomes necessary; therefore, the use of such a low reactiontemperature is economically disadvantageous. Meanwhile, when thereaction temperature is excessively high, although the polymerizationrate is improved, thermal decomposition of the polymer is also inducedduring the production, making it difficult to produce a polyester havinga high polymerization degree. Accordingly, the lower limit of thereaction temperature is usually not lower than 150° C., preferably notlower than 180° C., while the upper limit thereof is usually not higherthan 280° C., preferably not higher than 260° C.

As for the reaction time, when it is excessively short, the reactionproceeds insufficiently to yield a polyester having a low polymerizationdegree. The resulting polyester exhibits a low tensile elongation atbreak and has a large amount of terminal carboxyl group, which oftencauses considerable deterioration in the tensile elongation at break.Meanwhile, when the reaction time is excessively long, since themolecular weight of the resulting polyester is markedly reduced due tothermal decomposition, not only the tensile elongation at break may beimpaired, but also the amount of terminal carboxyl group, which affectsthe durability of polymer, may be increased due to thermaldecomposition. Accordingly, the lower limit of the reaction time isusually not shorter than 2 hours, while the upper limit thereof isusually not longer than 15 hours, preferably not longer than 8 hours,more preferably not longer than 6 hours.

In the present invention, with regard to the molar ratio of the diolcomponent and the aliphatic dicarboxylic acid component required forobtaining a polyester having a desired polymerization degree, thepreferred range thereof is variable depending on the purpose thereof andthe types of the starting materials; however, the lower limit of theamount of the diol component is, per 1 mol of acid component, usuallynot less than 0.8 mol, preferably not less than 0.9 mol, while the upperlimit thereof is usually not more than 3.0 mol, preferably not more than2.7 mol, particularly preferably not more than 2.5 mol.

Further, in the present invention, it is preferred that thepolycondensation reaction be carried out in the presence of apolymerization catalyst. The timing of adding the polymerizationcatalyst is not particularly restricted as long as it is before thepolycondensation reaction. The polymerization catalyst may be added atthe time of feeding the starting materials or at the start of pressurereduction.

Examples of the polymerization catalyst generally include thosecompounds other than hydrogen and carbon that contain a metal elementbelonging to the Groups 1 to 14 in the periodic table. Specific examplesthereof include organic group-containing compounds such as carboxylates,alkoxy salts, organic sulfonates and β-diketonates, which contain atleast one metal selected from the group consisting of titanium,zirconium, tin, antimony, cerium, germanium, zinc, cobalt, manganese,iron, aluminum, magnesium, calcium, strontium, sodium and potassium;inorganic compounds such as oxides and halides of the above-describedmetals; and mixtures of these compounds. Because of the above-describedreason, these catalyst components may be contained in the polyesterstarting material derived from a biomass resource. In this a case, thestarting material does not have to be particularly purified and may beused as-is in the form of a starting material containing a metal.However, depending on the polyester to be produced, there are caseswhere the lower is the content of a metal atom of the Group 1 such assodium or potassium in the polyester starting material, the easier is itto produce a polyester having a high polymerization degree. In such acase, a starting material which is purified to such an extent that itdoes not substantially contain a metal element of the Group 1 issuitably employed.

Among the above-described polymerization catalysts, metal compoundscontaining titanium, zirconium, germanium, zinc, aluminum, magnesium orcalcium and mixtures of these metal compounds are preferred. Thereamong,titanium compounds, zirconium compounds and germanium compounds areparticularly preferred. Further, the catalyst is preferably in the formof a liquid or a compound soluble to an ester low polymer or polyesterat the time of polymerization because the polymerization rate isincreased when the catalyst is in a molten or dissolved state at thetime of polymerization.

As the titanium compound, tetraalkyl titanate is preferred, and specificexamples thereof include tetra-n-propyl titanate, tetraisopropyltitanate, tetra-n-butyl titanate, tetra-t-butyl titanate, tetraphenyltitanate, tetracyclohexyl titanate, tetrabenzyl titanate and mixedtitanates thereof. In addition, titanium (oxy)acetylacetonate, titaniumtetraacetylacetonate, titanium (diisopropoxide)acetylacetonate, titaniumbis(ammonium lactate)dihydroxide, titaniumbis(ethylacetoacetate)diisopropoxide, titanium(triethanolaminate)isopropoxide, polyhydroxytitanium stearate, titaniumlactate, titanium triethanolaminate, butyl titanate dimer and the likecan be suitably used as well. Moreover, titanium oxide and complexoxides containing titanium and silicon can also be suitably used.

Among these titanium compounds, tetra-n-propyl titanate, tetraisopropyltitanate, tetra-n-butyl titanate, titanium (oxy)acetylacetonate,titanium tetraacetylacetonate, titanium bis(ammoniumlactate)dihydroxide, polyhydroxytitanium stearate, titanium lactate,butyl titanate dimer, titanium oxide and titania/silica complex oxidesare preferred, and tetra-n-butyl titanate, titanium(oxy)acetylacetonate, titanium tetraacetylacetonate, polyhydroxytitaniumstearate, titanium lactate, butyl titanate dimer and titania/silicacomplex oxides are more preferred. Further, tetra-n-butyl titanate,polyhydroxytitanium stearate, titanium (oxy)acetylacetonate, titaniumtetraacetylacetonate and titania/silica complex oxides are particularlypreferred.

Specific examples of the zirconium compounds include zirconiumtetraacetate, zirconium acetate hydroxide, zirconiumtris(butoxy)stearate, zirconyl diacetate, zirconium oxalate, zirconyloxalate, zirconium potassium oxalate, polyhydroxyzirconium stearate,zirconium ethoxide, zirconium tetra-n-propoxide, zirconiumtetraisopropoxide, zirconium tetra-n-butoxide, zirconiumtetra-t-butoxide, zirconium tributoxyacetylacetonate and mixturesthereof. Further, zirconium oxide and complex oxides containingzirconium and silicon can also be suitably used.

Among these zirconium compounds, zirconyl diacetate, zirconiumtris(butoxy)stearate, zirconium tetraacetate, zirconium acetatehydroxide, zirconium ammonium oxalate, zirconium potassium oxalate,polyhydroxyzirconium stearate, zirconium tetra-n-propoxide, zirconiumtetraisopropoxide, zirconium tetra-n-butoxide and zirconiumtetra-t-butoxide are preferred, and zirconyl diacetate, zirconiumtetraacetate, zirconium acetate hydroxide, zirconiumtris(butoxy)stearate, zirconium ammonium oxalate, zirconiumtetra-n-propoxide and zirconium tetra-n-butoxide are more preferred.Further, zirconium tris(butoxy)stearate is particularly preferred sinceit allows a colorless polyester having a high polymerization degree tobe easily obtained.

Specific examples of the germanium compounds include inorganic germaniumcompounds such as germanium oxide and germanium chloride; and organicgermanium compounds such as tetraalkoxygermanium. From the standpointsof price, availability and the like, for example, germanium oxide,tetraethoxygermanium and tetrabutoxygermanium are preferred, andgermanium oxide is particularly preferred.

In cases where a metal compound is used as the polymerization catalyst,when the amount of the catalyst is excessively large, not only is iteconomically disadvantageous, but also the thermal stability of theresulting polymer is reduced. On the other hand, when the amount of thecatalyst is excessively small, the polymerization activity is decreased,so that the polymer becomes more likely to be decomposed during theproduction. Accordingly, the lower limit of the amount of the catalystto be used is, in terms of the amount of the metal with respect to thatof the resulting polyester, usually not less than 5 ppm, preferably notless than 10 ppm, while the upper limit thereof is usually not more than30,000 ppm, preferably not more than 1,000 ppm, more preferably not morethan 250 ppm, particularly preferably not more than 130 ppm.

<Polyester and Use Thereof>

A polyester produced by the method of the present invention is generallycharacterized in that the amount of terminal carboxylic acid, whichmarkedly deteriorates the thermal stability of a polymer, is small;therefore, the polyester has characteristic features that it hasexcellent thermal stability and a reduction in the quality at the timeof molding is thus small, that is, occurrence of side reactions such asbreakage of the terminal group and main chain is limited at the time ofmelt molding. Accordingly, in a preferred polyester produced in thepresent invention, the number of terminal COOH groups is, although itvaries depending on the polymerization degree of the polyester, usuallynot more than 100 equivalents/ton (hereinafter, may be abbreviated as“eq/ton”), preferably not more than 60 eq/ton, more preferably not morethan 40 eq/ton, particularly preferably not more than 30 eq/ton.Meanwhile, when the amount of terminal carboxyl group is excessivelysmall, the polymerization rate becomes extremely slow, so that a polymerhaving a high-polymerization degree cannot be produced. For this reason,the lower limit of the number of terminal COOH groups in the polyesteris usually not less than 0.1 eq/ton, more preferably not less than 1eq/ton.

It is usually preferred that the polyester produced in the presentinvention be one having limited coloration. The upper limit of theyellowness (Y.I. value) of the polyester is usually not higher than 50,preferably not higher than 30, more preferably not higher than 20, stillmore preferably not higher than 15, particularly preferably not higherthan 10. Meanwhile, the lower limit thereof is not particularlyrestricted; however, it is usually not lower than −20, preferably notlower than −10, more preferably not lower than −5, particularlypreferably not lower than −3, most preferably not lower than −1. Apolyester showing a high Y.I. value has a drawback in that the use of afilm, a sheet or the like thereof is restricted. Meanwhile, a polyestershowing a low Y.I. value is a more preferred embodiment; however, it iseconomically disadvantageous in that, for example, the production ofsuch a polymer requires complex production processes as well as anextremely large investment in the equipments. In the present invention,the Y.I. value is measured by the method according to JIS K7105.

In the polyester produced in the present invention, from the standpointof attaining practically sufficient mechanical characteristics, thereduced viscosity (ηsp/C) value is not less than 0.5, more preferablynot less than 1.6, still more preferably not less than 2.0, particularlypreferably not less than 2.3. From the standpoint of operability such asthe easiness of recovering the resulting polyester after thepolymerization reaction and the moldability thereof, the upper limit ofthe reduced viscosity (ηsp/C) value is usually not higher than 6.0,preferably not higher than 5.0, still more preferably not higher than4.0.

In the present invention, the reduced viscosity is measured under thefollowing measurement conditions.

[Conditions for Measuring Reduced Viscosity (ηsp/C)]

Viscosity tube: Ubbelohde's viscosity tube

Measuring temperature: 30° C.

Solvent: phenol/tetrachloroethane (1:1 weight ratio) solution

Polyester concentration: 0.5 g/d1

In the method of producing a polyester according to the presentinvention, a variety of additives, such as a heat stabilizer, anantioxidant, a nucleating agent, a flame retardant, an antistatic agent,a mold releasing agent and/or an ultraviolet absorber, may also be addedto the reaction system at the time of performing polymerization or tothe resulting polyester in such a range which does not adversely affectthe properties of the polyester.

Further, in addition to the above-described various additives, at thetime of molding, a reinforcing agent and a filler, such as a glassfiber, a carbon fiber, a titanium whisker, mica, talc, CaCO₃, TiO₂ andsilica, may also be added to perform molding.

The polyester obtained by the production method of the present inventionhas excellent heat resistance and color tone as well as excellenthydrolysis resistance and biodegradability and can be producedinexpensively; therefore, it is suitably used in a variety of filmapplications and applications of injection-molded articles.

A molded article can be produced by molding the polyester obtained bythe production method of the present invention. As a molding method, anyconventional method can be employed. Examples of the molded articlewhich can be obtained are shown below along with the applicationsthereof. Specific examples of the application include injection-moldedarticles (e.g., trays used for fresh foods, containers of fast foods andoutdoor leisure products), extrusion-molded articles (e.g., films,sheets, fishing lines, fishing nets, vegetation nets and water-holdingsheets) and blow-molded articles (e.g., bottles). In addition, thepolyester can also be utilized in, for example, agricultural films,coating materials, fertilizer coating materials, laminated films,plates, stretched sheets, monofilaments, multifilaments, nonwovenfabrics, flat yarns, staples, crimped fibers, striated tapes, splityarns, composite fibers, blow bottles, foamed articles, shopping bags,trash bags, compost bags, cosmetic containers, detergent containers,bleach containers, ropes, tying materials, surgical sutures, sanitarycover stock materials, cooler boxes, cushioning films and syntheticpapers.

EXAMPLES

The present invention will now be described in more detail by way ofexamples thereof; however, the present invention is not restrictedthereto.

<Method of Quantifying Organic Acid>

The amount of organic acid was measured by quantitative analysis usingHPLC under the following conditions.

Column: Ultron PS-80H (8.0 mm I.D.×300 mm), manufactured by ShinwaChemical Industries Ltd.

Eluent: water (perchloric acid) (60% aqueous perchloric acid solution,1.8 ml/1 L—H₂O)

Temperature: 60° C.

Detection: RI, UV (210 nm)

<Method of Quantifying Ammonium Ion>

The amount of ammonium ion was measured by quantitative analysis usingion chromatography under the following conditions.

Column: GL-IC-C75 (4.6 mm I.D.×150 mm)

Eluent: 3.5 mmol/L sulfuric acid

Column temperature: 40° C.

<Method of Quantifying Aromatic Carboxylic Acid>

In Experimental Examples 1 to 3, the amount of aromatic carboxylic acidwas measured by quantitative analysis using HPLC under the followingconditions. In Example 2 and Comparative Example 2, the measurement wascarried out under the same conditions as in the above-described methodof quantifying organic acid.

Column: Develosil C30-UG (3 μm, 4.6 mm I.D.×100 mm), manufactured byNomura Chemical Co., Ltd.

Eluent: 0.02% aqueous formic acid solution, 1.0 mL/min

Temperature: 40° C.

Detector: UV (280 nm)

<Polymer Evaluation Method>

Yellow Index (Hereinafter, May be Abbreviated as “Y.I.”)

In accordance with the method prescribed in JIS K7105, a chip of theobtained polymer was placed in a cell and the Y.I. thereof was measured4 times by a reflection method using Color Meter ZE-6000 (manufacturedby Nippon Denshoku Industries Co., Ltd.). The average value thereof wasused as the Y.I. value of the polymer.

Reduced Viscosity

The obtained polyester was dissolved in phenol/tetrachloroethane (1/1(mass ratio) mixture) to a concentration of 0.5 g/dl. Then, in a 30° C.thermostat bath of the resulting solution, the time (t: seconds)required for a viscosity tube to fall was measured. Further, the timerequired for the viscosity tube to fall in the bath of the solvent alone(t₀: seconds) was measured to calculate the reduced viscosity at 30° C.(η_(sp)/C=(t−t₀)/t₀·C; C represents the concentration of the solution).

Amount of Terminal Carboxyl Group

The obtained polyester was dissolved in benzyl alcohol and the resultingsolution was titrated with 0.1N NaOH to determine the amount of terminalcarboxyl group as an equivalent amount of terminal acid group per 1×10⁶g of the polyester.

Example 1 Preparation of Brevibacterium flavum MJ233/ΔQsuB/PC-4/ΔLDHPreparation of QsuB-Disrupted Strain

(A) Extraction of Genomic DNA from MJ233 Strain

In 10 mL of a seed culture medium [2 g of urea, 7 g of (NH₄)₂SO₄, 0.5 gof KH₂PO₄, 0.5 g of K₂HPO₄, 0.5 g of MgSO₄.7H₂O, 6 mg of FeSO₄.7H₂O, 6mg of MnSO₄.4-5H₂O, 200 μg of biotin, 100 μg of thiamine, 1 g of yeastextract, 1 g of casamino acid and 20 g of glucose; dissolved in 1 L ofdistilled water], the Brevibacterium flavum MJ-233 strain was cultureduntil the late stage of logarithmic growth phase, and the resultingbacterial cells were collected by centrifugation (10,000 g, 5 minutes).The thus obtained bacterial cells were suspended in 0.15 mL of a 10 mMNaCl/20 mM Tris buffer (pH 8.0)/1 mM EDTA.2Na solution containinglysozyme at a concentration of 10 mg/mL. Then, proteinase K was added tothe thus obtained suspension to a final concentration of 100 μg/mL andthe resultant was incubated at 37° C. for 1 hour. Thereafter, sodiumdodecyl sulfate was further added to a final concentration of 0.5% andthe resultant was incubated at 50° C. for 6 hours to performbacteriolysis. To the resulting lysate, an equivalent amount of aphenol/chloroform solution was added. After gently shaking the resultantat room temperature for 10 minutes, the whole amount thereof wascentrifuged (5,000 G, 20 minutes, 10 to 12° C.) and a supernatantfraction was recovered. Then, after adding sodium acetate to the thusrecovered fraction to a concentration of 0.3 M, a double amount ofethanol was further added and the resultant was mixed. The resultingmixture was centrifuged (15,000 G, 2 minutes) to recover precipitates,which were then washed with 70% ethanol and air-dried. To the thusobtained DNA, 5 mL of a 10 mM Tris buffer (pH 7.5)/1 mM EDTA.2Nasolution was added, and the resultant was left to stand at 4° C.overnight and then used as a template DNA in the later PCR.

(B) Construction of Plasmid for Disruption of QsuB Gene

A DNA fragment of the qsuB gene originated from the Brevibacteriumflavum MJ233 strain in which an internal sequence thereof was deletedwas obtained by performing crossover PCR using the DNA prepared in theabove-described (A) as a template and synthetic DNAs (SEQ ID NOs:1, 2, 3and 4) which were designed based on a sequence of the vicinity of theqsuB gene of the Corynebacterium glutamicum ATCC13032 strain whoseentire genomic sequence has been reported (GenBank Accession No.BA000036). PCR of the DNA fragment containing the 5′-end region of theqsuB gene was performed using the synthetic DNAs shown in SEQ ID NOs:1and 2 as primers, while PCR of the DNA fragment containing the 3′-endregion of the qsuB gene was performed using the synthetic DNAs shown inSEQ ID NOs:3 and 4 as primers. The composition of the reaction solutionwas as follows: 1 μL of the template DNA, 0.5 μL of PfxDNA polymerase(manufactured by Invitrogen Corporation), 1-fold concentration of theattached buffer, 0.4 μM of each primer, 1 mM MgSO₄ and 0.2 μM dNTPs weremixed and the total volume was adjusted to 50 μL. The reactiontemperature conditions were as follows: a DNA thermal cycler (PTC-200,manufactured by MJ Research Inc.) was used and a cycle of 94° C. for 15seconds, 55° C. for 30 seconds and 68° C. for 45 seconds was repeated 35times. It is noted here, however, that the reaction solution wasretained at 94° C. for 2 minutes in the first cycle and at 68° C. for 3minutes in the final cycle. Then, PCR was further carried out using theresulting two amplification products as templates and the synthetic DNAsshown in SEQ ID NOs:1 and 4 as primers. Here, the composition of thereaction solution was as follows: 1 μL of the template DNAs, 0.5 μL ofPfxDNA polymerase (manufactured by Invitrogen Corporation), 1-foldconcentration of the attached buffer, 0.4 μM of each primer, 1 mM MgSO₄and 0.2 μM dNTPs were mixed and the total volume was adjusted to 50 μL.The reaction temperature conditions were as follows: a DNA thermalcycler (PTC-200, manufactured by MJ Research Inc.) was used and a cycleof 94° C. for 15 seconds, 55° C. for 30 seconds and 68° C. for 1 minuteand 20 seconds was repeated 35 times. It is noted here, however, thatthe reaction solution was retained at 94° C. for 2 minutes in the firstcycle and at 68° C. for 3 minutes in the final cycle. The thus obtainedDNA fragment in which the internal sequence of the qsuB gene was deletedwas purified using ChargeSwitch PCR Clean-Up Kit (manufactured byInvitrogen Corporation) and then digested with restriction enzymes XhoIand SacI. After separating the resulting DNA fragment of about 1.2 kb by0.9% agarose gel electrophoresis (SeaKem GTG agarose; manufactured byFMC BioProducts), the DNA fragment was detected by visualizing it withethidium bromide staining and then recovered from the gel usingZymoclean Gel DNA Recovery Kit (manufactured by Zymo ResearchCorporation). The thus obtained DNA was mixed with a DNA prepared bydigesting the plasmid pKMB1 (JP 2005-95169A) with restriction enzymesXhoI and SacI, and these DNAs were ligated with each other usingLigation Kit ver. 2 (manufactured by Takara Bio Inc.). Using the thusobtained plasmid DNA, Escherichia coli (DH5α strain) was transformed andthen spread onto an LB agar medium containing 50 μg/mL of kanamycin and50 μg/mL of X-Gal. A clone which formed a white colony on this mediumwas cultured in a liquid medium by a conventional method and a plasmidDNA was then purified. The thus obtained plasmid DNA was digested withrestriction enzymes XhoI and SacI. As a result, an insert fragment ofabout 1.2 kb was detected and this was named “pQsuB1”. The constructionprocess of the pQsuB1 is shown in FIG. 3.

(C) Preparation of QsuB-Disrupted Strain

As a sample strain for preparing a QsuB-disrupted strain, theBrevibacterium flavum MJ233/PC-4/ΔLDH strain prepared in thelater-described Reference Example 1 was employed. A plasmid DNA to beused for transformation of the Brevibacterium flavum MJ233/PC-4/ΔLDHstrain was prepared from Escherichia coli JM110 strain which wastransformed with the pQsuB1 plasmid constructed in the above-described(B). The transformation of the Brevibacterium flavum MJ233/PC-4/ΔLDHstrain was carried out by an electric pulse method (Vertes A. A., InuiM., Kobayashi M., Kurusu Y. and Yukawa H., Res. Microbiol., 1993, vol.144 (3), p 181-185), and the resulting transformant was spread onto anLBG agar medium containing 50 μg/mL of kanamycin [10 g of tryptone, 5 gof yeast extract, 5 g of NaCl, 20 g of glucose and 15 g of ager;dissolved in 1 L of distilled water]. In the strain that grew on thismedium, since the pQsuB1 is a plasmid which cannot be replicated in thecells of the Brevibacterium flavum MJ233 strain, as a result ofhomologous recombination between the qsuB gene of the plasmid and theqsuB gene on the genome of the Brevibacterium flavum MJ-233 strain, akanamycin-resistant gene originated from the plasmid is expected to havebeen inserted in the genome. Whether or not the kanamycin-resistantstrain obtained in this manner carries such gene derived by homologousrecombination between the qsuB gene existing on its genome and the qsuBgene existing on the plasmid pQsuB1 was verified by performing colonyPCR using the synthetic DNAs shown in SEQ ID NOs:1 and 5 and SEQ IDNOs:4 and 6 as primers. The template DNA was prepared in the form of asupernatant which was obtained by suspending the formed colonies in 50μL of sterilized water and boiling the resulting suspension for 5minutes. The composition of the reaction solution was as follows: 1 μLof the template DNA, 0.2 μL of Ex-Taq DNA polymerase (manufactured byTakara Bio Inc.), 1-fold concentration of the attached buffer, 0.2 μM ofeach primer and 0.2 μM dNTPs were mixed and the total volume wasadjusted to 20 μL. The reaction temperature conditions were as follows:a DNA thermal cycler (PTC-200, manufactured by MJ Research Inc.) wasused and a cycle of 98° C. for 10 seconds, 55° C. for 20 seconds and 72°C. for 2 minutes was repeated 35 times. It is noted here, however, thatthe reaction solution was retained at 95° C. for 2 minutes in the firstcycle and at 72° C. for 3 minutes in the final cycle. As a result ofanalyzing the kanamycin-resistant strain by the above-described method,a strain which yields a PCR amplification product of 1,256 bp with thecombination of SEQ ID NOs:1 and 5 and a PCR amplification product of1,866 bp with the combination of SEQ ID NOs:4 and 6 was selected, andthis strain was named “Brevibacterium flavum MJ233/ΔQsuB/PC-4/ΔLDH”.

(Evaluation of Growth of QsuB-Disrupted Strain on Synthetic Medium)

A MM medium [4 g of urea, 14 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄, 0.5 g ofK₂HPO₄, 0.5 g of MgSO₄.7H₂O, 20 mg of FeSO₄.7H₂O, 20 mg of MnSO₄.H₂O,200 μg of D-biotin and 200 μg of thiamine hydrochloride; dissolved in 1L of distilled water] in an amount of 100 mL was placed in a 500-mLErlenmeyer flask and heat-sterilized at 120° C. for 20 minutes. Themedium was then cooled to room temperature and 6 mL of 50% aqueousglucose solution which had been sterilized in advance and 50 μL offilter-sterilized 5% aqueous kanamycin solution were added thereto. TheBrevibacterium flavum MJ233/ΔQsuB/PC-4/ΔLDH strain was inoculated to theresulting medium to an absorbance (OD₆₆₀) of 1.0 and cultured at 30° C.with stirring at 160 rpm. At 1.5 hours, 3.1 hours, 5.0 hours, 7.0 hours,9.0 hours, 11.0 hours and 23.5 hours after the start of the culture, theabsorbance (OD₆₆₀) of the culture medium was measured. The measurementresults are shown in Table 1.

Reference Example 1 Preparation of Brevibacterium flavum MJ233/PC-4/ΔLDHStrain (Preparation of Pyruvate Carboxylase (PC)-Enhanced Strain)

(A) Extraction of Genomic DNA from Brevibacterium flavum MJ233 Strain

In 10 mL of A medium [2 g of urea, 7 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄,0.5 g of K₂HPO₄, 0.5 g of MgSO₄.7H₂O, 6 mg of FeSO₄.7H₂O, 6 mg ofMnSO₄.4-5H₂O, 200 μg of biotin, 100 μg of thiamine, 1 g of yeastextract, 1 g of casamino acid and 20 g of glucose; dissolved in 1 L ofdistilled water], Brevibacterium flavum MJ-233 strain was cultured untilthe late stage of logarithmic growth phase, and the resulting bacterialcells were collected by centrifugation (10,000 g, 5 minutes). The thusobtained bacterial cells were suspended in 0.15 mL of a 10 mM NaCl/20 mMTris buffer (pH 8.0)/1 mM EDTA.2Na solution containing lysozyme at aconcentration of 10 mg/mL. Then, proteinase K was added to the thusobtained suspension to a final concentration of 100 μg/mL and theresultant was incubated at 37° C. for 1 hour. Thereafter, sodium dodecylsulfate was further added to a final concentration of 0.5% and theresultant was incubated at 50° C. for 6 hours to perform bacteriolysis.To the resulting lysate, an equivalent amount of a phenol/chloroformsolution was added. After gently shaking the resultant at roomtemperature for 10 minutes, the whole amount thereof was centrifuged(5,000 G, 20 minutes, 10 to 12° C.) and a supernatant fraction wasrecovered. Then, after adding sodium acetate to the thus recoveredfraction to a concentration of 0.3 M, a double amount of ethanol wasfurther added and the resultant was mixed. The resulting mixture wascentrifuged (15,000 G, 2 minutes) to recover precipitates, which werethen washed with 70% ethanol and air-dried. To the thus obtained DNA, 5mL of a 10 mM Tris buffer (pH 7.5)/1 mM EDTA.2Na solution was added, andthe resultant was left to stand at 4° C. overnight and then used as atemplate DNA in the later PCR.

(B) Construction of Plasmid for Substitution of PC Gene Promoter

A DNA fragment of the N-terminal region of the pyruvate carboxylase geneoriginated from the Brevibacterium flavum MJ233 strain was obtained byperforming PCR using the DNA prepared in the above-described (A) as atemplate and synthetic DNAs (SEQ ID NOs:19 and 20) which were designedbased on the sequence of the pyruvate carboxylase gene of theCorynebacterium glutamicum ATCC 13032 strain whose entire genomicsequence has been reported (GenBank Accession No. BA000036). It is notedhere that the DNA shown in SEQ ID NO:19 was phosphorylated at the5′-end. The composition of the reaction solution was as follows: 1 μL ofthe template DNA, 0.2 μL of PfxDNA polymerase (manufactured byInvitrogen Corporation), 1-fold concentration of the attached buffer,0.3 μM of each primer, 1 mM MgSO₄ and 0.25 μM dNTPs were mixed and thetotal volume was adjusted to 20 μL. The reaction temperature conditionswere as follows: a DNA thermal cycler (PTC-200, manufactured by MJResearch Inc.) was used and a cycle of 94° C. for 20 seconds, 60° C. for20 seconds and 72° C. for 1 minute was repeated 35 times. It is notedhere, however, that the reaction solution was retained at 94° C. for 1minute and 20 seconds in the first cycle and at 72° C. for 4 minutes inthe final cycle. The resulting amplification products were verified byseparating them by 0.75% agarose gel electrophoresis (SeaKem GTGagarose; manufactured by FMC BioProducts) and then visualizing withethidium bromide staining. As a result, a fragment of about 0.9 kb wasdetected. The DNA fragment of interest was recovered from the gel usingQIAQuick Gel Extraction Kit (manufactured by QIAGEN) as a PC geneN-terminal fragment.

Meanwhile, a TZ4 promoter fragment which is originated from theBrevibacterium flavum MJ233 strain and constitutively highly expressedwas prepared by performing PCR using the plasmid pMJPC1 (JP 2005-95169A)as a template and the synthetic DNAs shown in SEQ ID NOs:21 and 22. Itis noted here that the DNA shown in SEQ ID NO:22 was phosphorylated atthe 5′-end. The composition of the reaction solution was as follows: 1μL of the template DNA, 0.2 μL of PfxDNA polymerase (manufactured byInvitrogen Corporation), 1-fold concentration of the attached buffer,0.3 μM of each primer, 1 mM MgSO₄ and 0.25 μM dNTPs were mixed and thetotal volume was adjusted to 20 μL. The reaction temperature conditionswere as follows: a DNA thermal cycler (PTC-200, manufactured by MJResearch Inc.) was used and a cycle of 94° C. for 20 seconds, 60° C. for20 seconds and 72° C. for 30 seconds was repeated 25 times. It is notedhere, however, that the reaction solution was retained at 94° C. for 1minute and 20 seconds in the first cycle and at 72° C. for 3 minutes inthe final cycle. The resulting amplification products were verified byseparating them by 1.0% agarose gel electrophoresis (SeaKem GTG agarose;manufactured by FMC BioProducts) and then visualizing with ethidiumbromide staining. As a result, a fragment of about 0.5 kb was detected.The DNA fragment of interest was recovered from the gel using QIAQuickGel Extraction Kit (manufactured by QIAGEN) as a TZ4 promoter fragment.

The thus obtained PC gene N-terminal fragment and TZ4 promoter fragmentwere mixed and ligated with each other using Ligation Kit ver. 2(manufactured by Takara Shuzo Co., Ltd.). Then, the resultant wasdigested with a restriction enzyme PstI and the resulting fragments wereseparated by 1.0% agarose gel electrophoresis (SeaKem GTG agarose;manufactured by FMC BioProducts). A DNA fragment of about 1.0 kb wasrecovered by using QIAQuick Gel Extraction Kit (manufactured by QIAGEN)as TZ4 promoter::PC gene N-terminal fragment. Further, this DNA fragmentwas mixed with a DNA prepared by digesting Escherichia coli plasmidpHSG299 (manufactured by Takara Shuzo Co., Ltd.) with PstI and they wereligated with each other using Ligation Kit ver. 2 (manufactured byTakara Shuzo Co., Ltd.). Using the thus obtained plasmid DNA,Escherichia coli (DH5α strain) was transformed. The recombinantEscherichia coli obtained in this manner was then spread onto an LB agarmedium containing 50 μg/mL of kanamycin and 50 μg/mL of X-Gal. A clonewhich formed a white colony on this medium was cultured in a liquidmedium by a conventional method and a plasmid DNA was then purified. Thethus obtained plasmid DNA was digested with a restriction enzyme PstI.As a result, an insert fragment of about 1.0 kb was detected and thisplasmid was named “pMJPC17.1”.

A DNA fragment of a 5′-upstream region of the pyruvate carboxylase geneoriginated from the Brevibacterium flavum MJ233 strain was obtained byperforming PCR using the DNA prepared in the above-described (A) as atemplate and synthetic DNAs (SEQ ID NOs:23 and 24) which were designedbased on the sequence of the pyruvate carboxylase gene of theCorynebacterium glutamicum ATCC13032 strain whose entire genomicsequence has been reported (GenBank Accession No. BA000036). Thecomposition of the reaction solution was as follows: 1 μL of thetemplate DNA, 0.2 μL of PfxDNA polymerase (manufactured by InvitrogenCorporation), 1-fold concentration of the attached buffer, 0.3 μM ofeach primer, 1 mM MgSO₄ and 0.25 μM dNTPs were mixed and the totalvolume was adjusted to 20 μL. The reaction temperature conditions wereas follows: a DNA thermal cycler (PTC-200, manufactured by MJ ResearchInc.) was used and a cycle of 94° C. for 20 seconds, 60° C. for 20seconds and 72° C. for 30 seconds was repeated 35 times. It is notedhere, however, that the reaction solution was retained at 94° C. for 1minute and 20 seconds in the first cycle and at 72° C. for 5 minutes inthe final cycle. The resulting amplification products were verified byseparating them by 1.0% agarose gel electrophoresis (SeaKem GTG agarose;manufactured by FMC BioProducts) and then visualizing with ethidiumbromide staining. As a result, a fragment of about 0.7 kb was detected.The DNA fragment of interest was recovered from the gel using QIAQuickGel Extraction Kit (manufactured by QIAGEN). The thus recovered DNAfragment was phosphorylated at the 5′-end with T4 Polynucleotide Kinase(manufactured by Takara Shuzo Co., Ltd.) and then ligated to the SmaIsite of the Escherichia coli vector pUC 119 (manufactured by TakaraShuzo Co., Ltd.) using Ligation Kit ver. 2 (manufactured by Takara ShuzoCo., Ltd.). Using the thus obtained plasmid DNA, Escherichia coli (DH5αstrain) was transformed. The recombinant Escherichia coli obtained inthis manner was then spread onto an LB agar medium containing 50 μg/mLof ampicillin and 50 μg/mL of X-Gal. A clone which formed a white colonyon this medium was cultured in a liquid medium by a conventional methodand a plasmid DNA was then purified. Thereafter, the thus obtainedplasmid DNA was subjected to PCR using the synthetic DNAs shown in SEQID NOs:25 and 24 as primers. The composition of the reaction solutionwas as follows: 1 ng of the above-described plasmid, 0.2 μL of Ex-TaqDNA polymerase (manufactured by Takara Shuzo Co., Ltd.), 1-foldconcentration of the attached buffer, 0.2 μM of each primer and 0.25 μMdNTPs were mixed and the total volume was adjusted to 20 μL. Thereaction temperature conditions were as follows: a DNA thermal cycler(PTC-200, manufactured by MJ Research Inc.) was used and a cycle of 94°C. for 20 seconds, 60° C. for 20 seconds and 72° C. for 50 seconds wasrepeated 20 times. It is noted here, however, that the reaction solutionwas retained at 94° C. for 1 minute and 20 seconds in the first cycleand at 72° C. for 5 minutes in the final cycle. As a result of verifyingthe presence or absence of inserted DNA fragment in this manner, aplasmid which was found to contain an amplification product of about 0.7kb was selected and named “pMJPC5.1”.

Then, the thus obtained plasmids pMJPC17.1 and pMJPC5.1 were eachdigested with an enzyme XbaI. Thereafter, the resultants were mixed andligated using Ligation Kit ver. 2 (manufactured by Takara Shuzo Co.,Ltd.). Then, the resultant was digested with restriction enzymes SacIand SphI and the resulting fragments were separated by 0.75% agarose gelelectrophoresis (SeaKem GTG agarose; manufactured by FMC BioProducts). ADNA fragment of about 1.75 kb was recovered by using QIAQuick GelExtraction Kit (manufactured by QIAGEN). The thus obtained DNA fragment,in which the TZ4 promoter was inserted between the 5′-upstream regionand the N-terminal region of the PC gene, was mixed with a DNA preparedby digesting the plasmid pKMB1 (JP 2005-95169A) containing sacB genewith restriction enzymes SacI and SphI, and these DNAs were ligated witheach other using Ligation Kit ver. 2 (manufactured by Takara Shuzo Co.,Ltd.). Using the thus obtained plasmid DNA, Escherichia coli (DH5αstrain) was transformed. The recombinant Escherichia coli obtained inthis manner was then spread onto an LB agar medium containing 50 μg/mLof kanamycin and 50 μg/mL of X-Gal. A clone which formed a white colonyon this medium was cultured in a liquid medium by a conventional methodand a plasmid DNA was then purified. The thus obtained plasmid DNA wasdigested with restriction enzymes SacI and SphI. As a result, an insertfragment of about 1.75 kb was detected and this plasmid was named“pMJPC17.2” (FIG. 2).

(C) Preparation of PC-Enhanced Strain

A plasmid DNA to be used for transformation of the Brevibacterium flavumMJ233/ΔLDH (lactate dehydrogenase gene-disrupted strain: JP 2005-95169A)was prepared once again from Escherichia coli JM110 strain which wastransformed with the plasmid DNA of pMJPC17.2 by a calcium chloridemethod (Journal of Molecular Biology, 53, p 159, 1970). Thetransformation of the Brevibacterium flavum MJ233/ΔLDH strain wascarried out by an electric pulse method (Res. Microbiol., vol. 144, p.181-185, 1993), and the resulting transformant was spread onto an LBGagar medium containing 25 μg/mL of kanamycin [10 g of tryptone, 5 g ofyeast extract, 5 g of NaCl, 20 g of glucose and 15 g of ager; dissolvedin 1 L of distilled water]. In the strain that grew on this medium,since the pMJPC17.2 is a plasmid which cannot be replicated in the cellsof the Brevibacterium flavum MJ233 strain, as a result of homologousrecombination between the PC gene of the plasmid and the PC gene on thegenome of the Brevibacterium flavum MJ233 strain, a kanamycin-resistantgene and a sacB gene that are originated from the plasmid are expectedto have been inserted in the genome. Then, the above-describedhomologous recombinant strain was cultured in a liquid LBG mediumcontaining 25 μg/mL of kanamycin. This culture solution was then, in anamount corresponding to about 1,000,000 cells, spread onto 10%sucrose-containing LBG medium. As a result, several tens of strains,which were believed to have become sucrose-insensitive due to the lossof sacB gene caused by the second homologous recombination, wereobtained. These strains obtained in this manner included those in whichthe TZ4 promoter originated from pMJPC17.2 was inserted in the upstreamof the PC gene, as well as those which were converted back to be of awild-type. Whether the PC gene is of a promoter-substituted type or awild type can be easily verified by directly subjecting cells obtainedby culturing in a liquid LBG medium to PCR and then detecting the PCgene. When the TZ4 promoter and the PC gene are analyzed by using theprimers for PCR amplification (SEQ ID NOs: 26 and 27), a DNA fragment of678 bp should be observed for the promoter-substituted type. As a resultof analyzing the strain which was transformed to be sucrose-insensitiveby the above-described method, a strain inserted with the TZ4 promoterwas selected and this strain was named “Brevibacterium flavumMJ233/PC-4/ΔLDH”.

(D) Measurement of Pyruvate Carboxylase Activity

The transformed Brevibacterium flavum MJ233/PC-4/ΔLDH strain obtained inthe above-described (C) was cultured overnight in 100 mL of A mediumcontaining 2% glucose. After recovering the resulting bacterial cells,the cells were washed with 50 mL of 50 mM potassium phosphate buffer (pH7.5) and then resuspended in 20 mL of buffer having the samecomposition. This suspension was homogenized by using Sonifier 350(manufactured by Branson Ultrasonics Corporation) and centrifuged toobtain a supernatant as a cell-free extract. Using the thus obtainedcell-free extract, the activity of pyruvate carboxylase was measured.The measurement of the enzyme activity was carried out by allowing areaction to take place at 25° C. in a reaction solution containing 100mM Tris/HCl buffer (pH 7.5), 0.1 mg/10 ml biotin, 5 mM magnesiumchloride, 50 mM sodium bicarbonate, 5 mM sodium pyruvate, 5 mM sodiumadenosine triphosphate, 0.32 mM NADH, 20 units/1.5 ml malatedehydrogenase (manufactured by Wako Pure Chemical Industries, Ltd.;originated from yeast) and the enzyme. Here, “1 U” was defined as anamount of the enzyme required for catalyzing a 1-μmol reduction of NADHin a period of 1 minute. The cell-free extract in which the expressionof pyruvate carboxylase was enhanced had a specific activity of 0.1U/mg-protein. It is noted here that the cells obtained by culturing theparent strain, MJ233/ΔLDH strain, in the same manner had a specificactivity of below the detection limit of this enzyme activity measuringmethod.

Comparative Example 1 Evaluation of Growth on Synthetic Medium

The evaluation was carried out in the same manner as in Example 1 exceptthat the Brevibacterium flavum MJ233/PC-4/ΔLDH strain was used in placeof the Brevibacterium flavum MJ233/ΔQsuB/PC-4/ΔLDH strain. It is notedhere that kanamycin was not added during the culture. In the same manneras in Example 1, the absorbance (OD₆₆₀) of the culture medium wasmeasured at 1.5 hours, 3.1 hours, 5.0 hours, 7.0 hours, 9.0 hours, 11.0hours and 23.5 hours after the start of the culture. The measurementresults are shown in Table 1.

TABLE 1 Culturing time Example 1 Comparative Example 1 [h] OD₆₆₀ OD₆₆₀1.5 1.16 1.30 3.1 1.82 2.03 5.0 2.98 3.42 7.0 4.55 4.85 9.0 6.88 7.2011.0 8.27 8.29 23.5 9.01 8.75

From the results shown in Table 1, it was confirmed that the disruptionof the qsuB gene does not adversely affect the growth on the syntheticmedium.

Example 2 Production of Succinic Acid by QsuB-Disrupted Strain

A medium [4 g of urea, 14 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄, 0.5 g ofK₂HPO₄, 0.5 g of MgSO₄.7H₂O, 20 mg of FeSO₄.7H₂O, 20 mg of MnSO₄.H₂O,200 μg of D-biotin, 200 μg of thiamine hydrochloride, 1 g of yeastextract and 1 g of casamino acid; dissolved in 1 L of distilled water]in an amount of 100 mL was placed in a 500-mL Erlenmeyer flask andheat-sterilized at 120° C. for 20 minutes. The medium was then cooled toroom temperature and 8 mL of 50% aqueous glucose solution which had beensterilized in advance and 50 μL of filter-sterilized 5% aqueouskanamycin solution were added thereto. The QsuB-disrupted strain(Brevibacterium flavum MJ233/ΔQsuB/PC-4/ΔLDH) prepared in Example 1 wasinoculated to the resulting medium to an absorbance (OD₆₆₀) of 1.0 andcultured at 30° C.

At 12 hours after the start of the culture, 3.16 g of ammoniumbicarbonate was added and allowed to react with the cells with the mouthof the Erlenmeyer flask being tightly closed with a parafilm. After thereaction, the cells were removed by centrifugation at 10,000 G for 5minutes and the organic acid concentration of the resulting supernatantwas analyzed by the above-described measurement method. As a result, theamount of protocatechuic acid generated in the culture medium after thereaction was found to be 12 ppm with respect to the amount of thegenerated succinic acid. The succinic acid concentration of the culturemedium after the reaction was 14.3 g/L.

Comparative Example 2

The evaluation was carried out in the same manner as in Example 2 exceptthat the pyruvate carboxylase (PC)-enhanced strain (Brevibacteriumflavum MJ233/PC-4/ΔLDH) prepared in Reference Example 1 was used inplace of the QsuB-disrupted strain (Brevibacterium flavumMJ233/ΔQsuB/PC-4/ΔLDH) prepared in Example 1. It is also noted here thatkanamycin was not added during the culture.

As a result of analyzing the organic acid concentration of the resultingculture medium in the same manner as in Example 2, the amount ofprotocatechuic acid generated in the culture medium after the reactionwas found to be 564 ppm with respect to the amount of the generatedsuccinic acid. The succinic acid concentration of the culture mediumafter the reaction was 14.5 g/L.

From these results, it was confirmed that the disruption of the qsuBgene markedly reduces the by-production of protocatechuic acid.

Experimental Example 1 Model Experiment: Production of Polymer UsingSuccinic Acid Obtained in Comparative Example 2 (Preparation of SuccinicAcid-Containing Solution A)

A food additive-grade succinic acid (manufactured by Kawasaki KaseiChemical Ltd.) and protocatechuic acid (manufactured by Wako PureChemical Industries, Ltd.) were dissolved in hot water of 80° C. toprepare a solution A having a succinic acid concentration of 35% byweight and a protocatechuic acid concentration of 197 ppm (succinicacid: 564 ppm).

(Crystallization)

The thus obtained succinic acid-containing solution A was stored in anaqueous succinic acid solution feed tank. The succinic acid-containingsolution A was then fed to a crystallization bath, whose jackettemperature was controlled at 80° C. by a program-controlled circulationthermostat bath, by means of a stock solution feed pump until the liquidlevel reached a prescribed point. Once the liquid level reached theprescribed point, while stirring the solution with paddle blades at arate of 500 rotation/minutes, the hot water being supplied to the jacketof the crystallization bath was cooled to 20° C. over a period of aboutone hour, thereby lowering the temperature inside the crystallizationbath to 20° C. After the crystallization bath was cooled to 20° C.,while maintaining this temperature, the stirring was continued foranother one hour.

Thereafter, while controlling the temperature of the cold water beingpassed through the jacket so that the temperature inside thecrystallization bath was maintained at 20° C., the succinicacid-containing solution A was continuously fed at a rate of 250ml/minute and a solid succinic acid-containing slurry was intermittentlyremoved into a slurry recovery tank once every 15 minutes or so in orderto keep the volume of the succinic acid-containing slurry in thecrystallization bath almost constant. The removed succinicacid-containing slurry was vacuum-filtered to be separated into asuccinic acid wet cake and a crystallization mother liquor every timethe slurry was removed.

This continuous crystallization operation was continued for 24 hours andsuccinic acid wet cakes that were obtained after 6 hours or later fromthe start of the continuous crystallization operation were recovered.The thus recovered succinic acid wet cakes were suspended and washed ina 5-times weight of 10° C. cold water. Thereafter, the resulting slurrywas vacuum-filtered to obtain a succinic acid wet cake. This wet cakewas then vacuum-dried at 80° C. to recover succinic acid.

The thus obtained succinic acid was dissolved in 80° C. hot water to asuccinic acid concentration of 35% by weight and the resulting solutionwas once again subjected to continuous crystallization for 7 hours bythe above-described crystallization operation. Succinic acid wet cakesthat were obtained between 6 and 7 hours in the continuouscrystallization operation were suspended and washed in a 5-times weightof 10° C. cold water. Thereafter, the resulting slurry wasvacuum-filtered to obtain a succinic acid wet cake. This wet cake wasthen vacuum-dried at 80° C. to recover succinic acid in an amount of 55g. As a result of quantifying the aromatic carboxylic acid contained inthe thus obtained succinic acid, it was found that the succinic acidcontained 3.6 ppm of protocatechuic acid.

(Production of Polymer)

To a reaction vessel equipped with a stirring device, a nitrogen inlet,a heating apparatus, a thermometer and a pressure reduction vent, 100parts by weight of the succinic acid obtained by the above-describedcrystallization operation was fed as a starting material along with 99.2parts by weight of industrial-grade 1,4-butanediol (manufactured byMitsubishi Chemical Corporation) and 0.38 parts by weight of malic acid(a total amount of 0.33 mol % with respect to the amount of the succinicacid), and the inside of the system was replaced to be a nitrogenatmosphere with nitrogen under reduced pressure. Then, with stirring,the inside of the system was heated to 230° C. over a period of 1 hourand then reaction was performed at this temperature for 1 hour. To thisreaction solution, a catalyst solution prepared by the below-describedmethod was added in such an amount that the amount of titanium atombecame 50 ppm with respect to the amount of the resulting polyestertheoretically calculated based on the amount of the starting materialused.

To a 500-cm³ eggplant-shaped glass flask equipped with a stirringdevice, 62.0 g of magnesium acetate tetrahydrate was placed and 250 g ofanhydrous ethanol (purity: not less than 99% by weight) was addedthereto. Further, 35.8 g of ethyl acid phosphate (mixed weight ratio ofmonoester body and diester body=45:55) was added and the resultingmixture was stirred at 23° C. After 15 minutes, complete dissolution ofmagnesium acetate was confirmed and then 75.0 g of tetra-n-butyltitanate was added. The resulting mixture was stirred for another 10minutes to obtain a uniform mixed solution. The thus obtained mixedsolution was transferred to a 1,000-cm³ eggplant-shaped flask, which wasthen placed in a 60° C. oil bath to concentrate the mixed solution underreduced pressure using an evaporator. After 1 hour, most of ethanol wasdistilled out, and a semi-transparent viscous liquid was remained. Then,the temperature of the oil bath was raised to 80° C. to furtherconcentrate the semi-transparent viscous liquid under a reduced pressureof 5 Ton. The viscous liquid gradually changed into the form of powderfrom the surface and completely powderized after 2 hours. Thereafter,the system was brought back to normal pressure using nitrogen and cooledto room temperature, thereby obtaining a pale yellow powder in an amountof 108 g. The thus obtained catalyst was subjected to metal elementanalysis and, as a result, it was found that the catalyst contained10.3% by weight of titanium atom, 6.8% by weight of magnesium atom and7.8% by weight of phosphorus atom and the molar ratios thereof were:T/P=0.77 and M/P=1.0. Furthermore, this powder-form catalyst wasdissolved in 1,4-butanediol so that the titanium atom content became34,000 ppm.

After adding the resulting catalyst solution, the inner temperature ofthe reaction vessel was slowly raised to 250° C. and, at the same time,the pressure was reduced to 0.06×10³ Pa over a period of 2 hours. Thereaction was allowed to proceed for 2.5 hours at this reduced pressure,thereby producing a polyester. When the thus obtained polyester wasevaluated in accordance with the above-described polymer evaluationmethod, it was found that the Y.I. value was 10, the reduced viscosity(ηsp/c) was 2.3 and the amount of terminal carboxyl group was 24 eq/ton.

Experimental Example 2 Model Experiment (1): Production of Polymer UsingSuccinic Acid Obtained in Example 2 (Preparation of SuccinicAcid-Containing Solution B, Crystallization Thereof and Production ofPolymer)

A food additive-grade succinic acid (manufactured by Kawasaki KaseiChemical Ltd.) and protocatechuic acid (manufactured by Wako PureChemical Industries, Ltd.) were dissolved in hot water of 80° C. toprepare a solution B having a succinic acid concentration of 35% byweight and a protocatechuic acid concentration of 4.2 ppm (succinicacid: 564 ppm).

By performing the crystallization operation in the same manner as inExperimental Example 1 except that the thus obtained succinicacid-containing solution B was used, 54 g of succinic acid wasrecovered. As a result of quantifying the aromatic carboxylic acidcontained in the thus obtained succinic acid, it was found that thesuccinic acid contained 0.1 ppm of protocatechuic acid.

Using the thus obtained succinic acid, a polyester was produced in thesame manner as in Experimental Example 1. When this polyester wasevaluated in accordance with the above-described polymer evaluationmethod, it was found that the Y.I. value was 5, the reduced viscosity(ηsp/c) was 2.3 and the amount of terminal carboxyl group was 24 eq/ton.

Experimental Example 3 Model Experiment (2): Production of Polymer UsingSuccinic Acid Obtained in Example 2 (Crystallization Using SuccinicAcid-Containing Solution B and Production of Polymer)

The above-described succinic acid-containing solution B was stored in anaqueous succinic acid solution feed tank and subjected to continuouscrystallization operation for 24 hours in the same manner as inExperimental Example 1. Succinic acid wet cakes that were obtained after6 hours or later from the start of the continuous crystallizationoperation were recovered. The thus recovered succinic acid wet cakeswere suspended and washed in a 5-times weight of 10° C. cold water.Thereafter, the resulting slurry was vacuum-filtered to obtain asuccinic acid wet cake. This wet cake was then vacuum-dried at 80° C. torecover succinic acid. As a result of quantifying the aromaticcarboxylic acid contained in the thus obtained succinic acid, it wasfound that the succinic acid contained 1 ppm of protocatechuic acid.

Using the thus obtained succinic acid, a polyester was produced in thesame manner as in Experimental Example 1. When this polyester wasevaluated in accordance with the above-described polymer evaluationmethod, it was found that the Y.I. value was 6, the reduced viscosity(ηsp/c) was 2.3 and the amount of terminal carboxyl group was 24 eq/ton.

Experimental Example 4 Preparation of Succinic Acid-Containing SolutionC, Crystallization Thereof and Production of Polymer

A food additive-grade succinic acid (manufactured by Kawasaki KaseiChemical Ltd.) and diammonium succinate (manufactured by Wako PureChemical Industries, Ltd.) were dissolved in hot water of 80° C. toprepare a solution C having a succinic acid concentration of 35% byweight and an ammonium ion concentration of 175 ppm (succinic acid:5,000 ppm).

By performing the crystallization operation in the same manner as inExperimental Example 1 except that the thus obtained succinicacid-containing solution C was used, 60 g of succinic acid wasrecovered. As a result of quantifying the ammonium ion contained in thethus obtained succinic acid, it was found that the succinic acidcontained ammonium ion in an amount of not higher than the detectionlimit (0.1 ppm).

Using the thus obtained succinic acid, a polyester was produced in thesame manner as in Experimental Example 1. When this polyester wasevaluated in accordance with the above-described polymer evaluationmethod, it was found that the Y.I. value was 5, the reduced viscosity(ηsp/c) was 2.3 and the amount of terminal carboxyl group was 24 eq/ton.

From the results of Experimental Examples 1 to 4, it is understood thatthe method of producing a polymer according to the present invention canreduce the coloration of resulting polymers.

Experimental Example 5

With reference to the method described in the below-listed References,uvitonic acid was synthesized. That is, 10 ml (2 mol) of anammonia-ethanol solution was placed in a 100-ml three-necked flask and,while stirring the solution with a magnetic stirrer under N₂, 0.91 g(0.01 mol) of pyruvic acid was added thereto dropwise at roomtemperature. The resulting solution slightly generated heat and whiteprecipitates were formed after a few minutes. Then, the thus obtainedwhite precipitates were washed with ethanol.

REFERENCES

-   1. J. Org. Chem., 50, 1688 (1985)-   2. Biochimie, 54, 115 (1972)-   3. Vegetable Physiology and Agriculture-   4. J. Org. Chem., 47, 1148 (1982)

Using a food additive-grade succinic acid (manufactured by KawasakiKasei Chemical Ltd.), polyesters were produced in the same manner as inthe above-described Experimental Example 1, except that protocatechuicacid (manufactured by Wako Pure Chemical Industries, Ltd.) or theuvitonic acid produced by the above-described method was added in theamount shown in Table 2. The Y.I. value of the thus obtained respectivepolyesters was measured in accordance with the above-described polymerevaluation method. The measurement results are shown in Table 2 below.

TABLE 2 Concentration of protocatechuic acid with respect to the amountof succinic acid at the time of feeding (ppm) 0 10 57 Y.I. value 5 17 40Concentration of uvitonic acid with respect to the amount of succinicacid at the time of feeding (ppm) 0 27 76 Y.I. value 5 10 17

From the above results, it is understood that protocatechuic acid anduvitonic acid, which are aromatic carboxylic acids, are substances thatcause coloration of a polymer.

INDUSTRIAL APPLICABILITY

In general, succinic acid is produced from a petrochemically-derivedmaterial and used in a wide variety of applications. For theseapplications, succinic acid derived from a bioresource can also bepreferably used in the same manner. For example, such succinic acid canbe used as: a raw material of 1,4-butanediol, 2-pyrrolidine,succinimide, maleic anhydride, itaconic acid, aspartic acid, maleicacid, fumaric acid, hydroxysuccinimide, maleimide, 4-aminobutyric acid,γ-aminobutyric acid, tetrahydrofuran, acrylic acid, succinic esters suchas dimethyl succinate and diethyl succinate, pyrrolidone,N-methylpyrrolidone and the like; as a starting material of polymercompounds such as polyester, polyurethane and polyamide and productsthereof; a food additive such as an acidulant, a flavoring agent, abrewing agent or a processed food additive; a bubble bath component; asynthetic material or component of pharmaceuticals and agriculturalchemicals such as plant growth inhibitors, herbicides, antibacterialagents, pesticides and mosquito attractants; a material or component ofmouthwash, cosmetics and the like; a material or component of productsthat are used for photographs and printings; a material or component ofadhesives and sealants such as high-temperature welding fluxes andalumite-treated surface adhesives; a material or component for metalprocessing such as powder nickel production, steel grinding bath, metalprocessing and washing solvents and binders for metal sintering; amaterial or component of solders and welding fluxes; a material orcomponent of auxiliary agents that are used in the production ofceramics, inorganics and the like, such as production of a poroustitanium oxide, production of boehmite, production of photocatalyticcoating agents and production of ceramics; a material or component ofdetergents and the like; a material or component of bleaches and thelike; a material or component of dyeing aids; a material or component ofelectrolyte solvents, plating solutions and the like; a material orcomponent of deodorants, air cleaning agents and the like; a material ofbioabsorbable compounds used for bioabsorbable surgical sutures and thelike; a material or component of treatment agents, softeners and thelike of textile goods; a material or component of fluxes, solvents andthe like; a material or component of water-soluble paint solvents; amaterial or component of biodegradable resins; a material or componentof sealants such as odor-free sealants; a material or component ofanticorrosive agents that are used in coating of steel products, copperproducts and alloy metal products, freeze proofing, metal processing,lead for perchloric acid, boiler water treatment and the like; amaterial or component for synthesizing lubricants such as syntheticlubricants, lubricants for heat-resistant plastics and electricalcontact lubricants; a material or component of solvent-removing washingagents and the like that used for resins, polymer materials and thelike; a material or component of products that are used in the textileindustry, dry cleaning and the like; a material or component ofpigments, dyes, inks and the like that are used in, for example, inksolvents, deinking agents, automobile top-coating agents, insulatingvarnishes, powder paints, inks for three-dimensional printing,photosetting-type paints, photosetting ink compositions, nanoparticleinks, inks for ink jet printers, printing screen washing agents, organicsemiconductor solutions, inks for color filter production, toners,quinacridone pigment production, succinyl succinate production and dyeintermediates; a material or component of oxygen-containing-type dieselfuels and the like; a material or component of cement admixtures, cementtreatment agents and the like; a material or component of enginecleaners and the like; a material or component of petroleum refinerysolvents and the like; a material or component of oil and natural gasextraction auxiliary agents such as proppant compositions and thoseauxiliary agents that are used for removal of precipitation filtercakes; a material or component of those products relating to natural gasproduction, such as natural gas dehydrating solvents; a material orcomponent of construction materials such as low-dust concrete flooringmaterials and asphalt pavement materials; and a material or component ofink solvents and deinking agents.

DESCRIPTION OF SEQUENCES

SEQ ID NO:1 (nucleotide sequence of a primer)SEQ ID NO:2 (nucleotide sequence of a primer)SEQ ID NO:3 (nucleotide sequence of a primer)SEQ ID NO:4 (nucleotide sequence of a primer)SEQ ID NO:5 (nucleotide sequence of a primer)SEQ ID NO:6 (nucleotide sequence of a primer)SEQ ID NOs:7 and 8 (nucleotide sequence of the aroF gene and amino acidsequence encoded thereby)SEQ ID NOs:9 and 10 (nucleotide sequence of the aroG gene and amino acidsequence encoded thereby)SEQ ID NOs:11 and 12 (nucleotide sequence of the aroB gene and aminoacid sequence encoded thereby)SEQ ID NOs:13 and 14 (nucleotide sequence of the qsuC gene and aminoacid sequence encoded thereby)SEQ ID NOs:15 and 16 (nucleotide sequence of the qsuB gene and aminoacid sequence encoded thereby)SEQ ID NOs:17 and 18 (nucleotide sequence of the qsuD gene and aminoacid sequence encoded thereby)SEQ ID NO:19 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)SEQ ID NO:20 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)SEQ ID NO:21 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)SEQ ID NO:22 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)SEQ ID NO:23 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)SEQ ID NO:24 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)SEQ ID NO:25 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)SEQ ID NO:26 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)SEQ ID NO:27 (nucleotide sequence of a polymer used in the constructionof pMJPC17.2)

1. A method of producing a polymer, which comprises the step of performing a polymerization reaction using, as a starting material, an organic acid obtained by allowing a microorganism or a treated cell of thereof to act on an organic raw material, wherein said microorganism has an ability to produce the organic acid and has been modified so as to produce less aromatic carboxylic acid as compared to an unmodified strain.
 2. The method according to claim 1, wherein said microorganism has been modified so that at least one enzyme activity selected from the group consisting of DAHP synthase activity, dehydroquinate synthase activity, dehydroquinate dehydratase activity and dehydroshikimate dehydratase activity is reduced as compared to an unmodified strain, and production of an aromatic carboxylic acid is thereby reduced.
 3. The method according to claim 1, wherein said organic acid is subjected to a crystallization treatment.
 4. The method according to claim 1, wherein said aromatic carboxylic acid is a hydroxybenzene carboxylic acid.
 5. The method according to claim 1, wherein said organic acid is succinic acid.
 6. The method according to claim 5, wherein said polymer is a polyester or a polyamide.
 7. A method of producing an organic acid, comprising the step of allowing a microorganism or a treated cell thereof to act on an organic raw material, wherein said microorganism has an ability to produce the organic acid and has been modified so as to produce less aromatic carboxylic acid as compared to an unmodified strain.
 8. The method according to claim 7, wherein said microorganism has been modified so that at least one enzyme activity selected from the group consisting of DAHP synthase activity, dehydroquinate synthase activity, dehydroquinate dehydratase activity and dehydroshikimate dehydratase activity is reduced as compared to an unmodified strain, and production of an aromatic carboxylic acid is thereby reduced.
 9. The method according to claim 7, wherein said microorganism or a treated cell thereof is allowed to act on said organic raw material in an anaerobic atmosphere.
 10. The method according to claim 7, wherein said organic acid is succinic acid.
 11. The method according to claim 7, further comprising the step of performing a crystallization treatment of said organic acid.
 12. The method according to claim 7, wherein said aromatic carboxylic acid is a hydroxybenzene carboxylic acid.
 13. The method according to claim 7, wherein said microorganism is at least one bacterium selected from the group consisting of coryneform bacteria, bacteria belonging to the genus Mycobacterium, bacteria belonging to the genus Rhodococcus, bacteria belonging to the genus Nocardia and bacteria belonging to the genus Streptomyces. 